Compositions and methods for treating obesity, obesity related disorders and for inhibiting the infectivity of human immunodeficiency virus

ABSTRACT

The present invention relates to methods and pharmaceutical compositions for treating obesity or obesity-related disorders in a subject suffering from or predisposed to developing obesity or an obesity-related disorder, or for inhibiting the infectivity of HIV, by administering oleuropein, an analogue or derivative thereof, or the major metabolites of oleuropein including oleuropein aglycone, hydroxytyrosol, and elenolic acid or their analogues, or derivatives thereof, an iridoid glycoside, or a secoiridoid glycoside or analogues or derivatives thereof, or any combination of the foregoing including olive leave extract. The invention also relates to methods for screening/diagnosing a subject having, or predisposed to having obesity or a related disorder by measuring the expression profiles of an adipogenic gene selected from PPARγ2, LPL and αP2 gene and gene product, or other adipogenic, lipogenic, or lipolytic genes and gene products in an individual. The invention further provides for screening for novel oleuropein analogues.

FIELD OF THE INVENTION

The present invention relates generally to methods and compositions formodulating the body weight of mammals including animals and humans, andmore particularly to materials identified herein as modulators ofweight, and the use of these materials for treating obesity anddisorders related to obesity and to the diagnostic and therapeutic usesto which such modulators may be put. The present invention also relatesto methods and compositions for inhibiting the infectivity of humanimmunodeficiency virus (HIV).

BACKGROUND OF THE INVENTION

Obesity, which is defined in general terms as an excess of body fatrelative to lean body mass, is now a world wide epidemic, and is one ofthe most serious contributors to increased morbidity and mortality.Obesity is prevalent in the United States, affecting more than 61% ofthe total population (Flegal, et al., Overweight and Obesity in theUnited States: Prevalence and Trends, 1960-1994. Int J Obes 22:39-47,1998). Obesity is defined more specifically by the United States Centersfor Disease Control and Prevention (CDC) as an excessively high amountof body fat or adipose tissue in relation to lean body mass andoverweight is defined as an increased body weight in relation to height,when compared to some standard of acceptable or desirable weight. TheCDC alternatively defines overweight as a person with a body mass index(BMI) between 25.0 and 29.9 and obesity is defined as a BMI greater thanor equal to 30.0. Obesity is often associated with psychological andmedical morbidities, the latter of which includes increased jointproblems, vascular diseases such as coronary artery disease,hypertension, stroke, and peripheral vascular disease. Obesity alsocauses metabolic abnormalities such as insulin resistance and Type IIdiabetes (non-insulin-dependent diabetes mellitus (NIDDM)),hyperlipidemia, and endothelial dysfunction. These abnormalitiespredispose the vasculature to injury, cellular proliferation and lipidoxidation, with resulting atherosclerosis leading to heart attack,stroke, and peripheral vascular diseases. In 1998, consumers spent $33billion in the United States for weight-loss products and services witha less than positive outcome (Serdula, et al., Prevalence of AttemptingWeight Loss and Strategies for Controlling Weight, JAMA 282:1353-1358,1999). Thus, obesity and its associated complications continue to be amajor problem throughout the worldwide health care system.

Obesity is clearly an important clinical problem with very broadreaching implications. There is a pressing need for more research on themolecular mechanisms that underlie obesity and its medical consequences,as well as new approaches for its treatment. To date, these approacheshave been limited to diet and exercise (therapeutic lifestyle changes),surgical procedures such as gastric bypass, and pharmacologic agents.Drug treatment for obesity has been disappointing since almost all drugtreatments for obesity are associated with undesirable side effects thatcontributed to their termination. A number of monoamines andneuropeptides are known to reduce food intake (Bray, et al.,Pharmacological Treatment of Obesity, Am J Clin Nutr 55:151S-319S,1992). Available pharmacotherapies have included Sibutramine (anappetite suppressant), Orlistat (a lipase inhibitor), fenfluramine anddexfenfluramine. Although body weight loss is effective, thesesympathomimetic drugs cause side effects including pulmonaryhypertension, neuroanatomic changes, and a typical valvular heartdiseases. For example, fenfluramine and dexfenfluramine were withdrawnfrom the market in 1997 because of associated cardiac valvulopathy(Connolly, et al., Valvular Heart Disease Associated WithFenfluramine-Phentermine, New Engl J Med 337-581-588, 1997). Thus,nutrition and dietary restriction are most desirable for weight loss.However, long-term success of dietary regulation is low because ofnoncompliance. The loss of motivation to change dietary habits necessaryto consume less fat and fewer calories results in regaining weight.

Thus, there are no real treatments based on the biology of the primarymetabolic abnormalities found in obesity and its related conditions,such as metabolic syndrome or atherosclerosis. Accordingly, there isstill a need for new compositions and methods that address treatingindividuals suffering from obesity and obesity-related disorders. Thereis also a need for new agents and compositions for treating individualsinfected with human immunodeficiency virus (HIV). It is toward thedevelopment of new compositions and methods for treating obesity andobesity-related disorders and for treating individuals infected with HIVthat the present invention is directed.

The citation of any reference herein should not be construed as anadmission that such reference is prior art to the instant invention.

SUMMARY OF THE INVENTION

In accordance with the broadest aspect of the invention, methods andcompositions comprising oleuropein, or an analogue or derivativethereof, or the major metabolites of oleuropein including oleuropeinaglycone, hydroxytyrosol, and elenolic acid or their analogues, orderivatives thereof, or an iridoid glycoside, or a secoiridoid glycosideor their analogues or derivatives thereof, or any combination of theforegoing including but not limited to olive leave extract, aredisclosed for treating obesity and obesity-related conditions ordisorders, as well as for inhibiting the infectivity of HIV. Inparticular, these compositions modulate adipogenesis, lipodystrophy,reduce fat accumulation and weight gain. These agents also prevent HIVviral fusion/entry into a host cell and bind the catalytic site of theHIV integrase. Thus, these agents provide an advantage over otheranti-viral therapies in that both viral entry and integration areinhibited. They exert their effect by modulating adipocytedifferentiation (adipogenesis), de-differentiation,transdifferentiation, and by decreasing the number of adipocytes (fatcells), or by modulating adipocyte metabolism (lipid synthesis, storage,accumulation, and utilization) so as to decrease fat accumulation anddecrease the size of the fat cell (decrease fat mass), increase fatburning and expenditure. These compositions also have an effect onadipogenic, lipogenic and lipolytic gene/gene product expression,perturbation of pre-adipocyte to adipocyte balance by promotingde-differentiation or transdifferentiation of adipocytes, the end resultbeing a reduction in fat accumulation (adipose mass) and a reduction inweight gain. Accordingly, these compositions are useful for treatingobesity and obesity-related disorders. In addition, these compositionshave been shown to reduce diet-induced atherogenesis, thus allowing fora means of treating one of the major conditions or disorders associatedwith, or resulting from, obesity. Moreover, the compositions disclosedmay also be utilized for treating other obesity-related disorders,including but not limited to, coronary artery disease, hypertension,stroke, peripheral vascular disease, insulin resistance, glucoseintolerance, diabetes mellitus, hyperlipidemia, atherosclerosis,cellular proliferation and endothelial dysfunction, diabeticdyslipidemia, HIV-related lipodystrophy, e.g. Highly ActiveAnti-Retroviral Therapy (HAART)-induced lipodystrophy, and metabolicsyndrome, type II diabetes, hyperinsulinemia, diabetic complicationsincluding diabetic neuropathy, nephropathy, retinopathy or cataracts,heart failure, hypercholesterolemia, inflammation, thrombosis,congestive heart failure, and any other cardiovascular disease relatedto obesity or an overweight condition, or obesity induced asthma, airwaydysfunction and pulmonary disorders. The compositions may also becontemplated for use in the production of lean meat from meat animals,e.g., beef cattle, lambs, hogs, chickens and turkeys.

Accordingly, a first aspect of the invention provides a method ofmodulating adipocyte differentiation or adipogenic gene expression,comprising administering a therapeutically effective amount ofoleuropein or an analogue, or derivative thereof, or oleuropein aglyconeor their analogues, or derivatives thereof, or hydrotyrosol, ordihydroxy phenol or their analogues, or derivatives thereof, or elenolicacid or their analogues, or derivatives thereof, or an iridoidglycoside, or a secoiridoid glycoside or their analogues, or derivativesthereof, or any combination of any of the foregoing thereof includingbut not limited to olive leave extract, to a mammalian subject in needthereof.

In one embodiment, oleuropein or its analogues, or derivatives thereof,or oleuropein aglycone or their analogues, or derivatives thereof, orhydrotyrosol, or dihydroxy phenol or their analogues, or derivativesthereof, or elenolic acid or their analogues, or derivatives thereof, oran iridoid glycoside, or a secoiridoid glycoside or their analogues, orderivatives thereof, or any combinations of any of the foregoingincluding but not limited to olive leave extract, up-regulates fatutilization, energy uncoupling, related regulators, factors and enzymesincluding but not limited to lipolytic genes/gene products in thesepathways.

In another embodiment, oleuropein or an analogue, or derivative thereof,or oleuropein aglycone or their analogues, or derivatives thereof, orhydrotyrosol, or dihydroxy phenol or their analogues, or derivativesthereof, or elenolic acid or their analogues, or derivatives thereof, oran iridoid glycoside, or a secoiridoid glycoside or their analogues, orderivatives thereof, or any combinations of any of the foregoingincluding but not limited to olive leave extract, blocks ordown-regulates adipocyte differentiation, regulators, factors, andenzymes involved in the adipogenic pathway. In yet another embodiment,oleuropein or an analogue, or derivative thereof or oleuropein aglyconeor their analogues, or derivatives thereof, or hydrotyrosol, ordihydroxy phenol or their analogues, or derivatives thereof, or elenolicacid or their analogues, or derivatives thereof, or an iridoidglycoside, or a secoiridoid glycoside or their analogues, or derivativesthereof, or any combinations of any of the foregoing thereof includingbut not limited to olive leave extract, enhances de-differentiation ofadipocytes and allows for transdifferentiation into osteoblasts, musclecells, cartilage, and bone as well as regulators and enzymes involved inthese pathways.

In yet another embodiment, the adipogenic gene whose expression ismodulated by oleuropein or an analogue, or derivative thereof, oroleuropein aglycone or their analogues, or derivatives thereof, orhydrotyrosol, or dihydroxy phenol or their analogues, or derivativesthereof, or elenolic acid or their analogues, or derivatives thereof, oran iridoid glycoside, or a secoiridoid glycoside or their analogues, orderivatives thereof, or any combinations of any of the foregoingincluding but not limited to olive leave extract, is selected from thegroup consisting of Peroxisome Proliferator-Activated Receptor γ(PPARγ), lipoprotein lipase (LPL) and the αP2 gene or gene product.Oleuropein or its analogues, or derivatives, or oleuropein aglycone ortheir analogues, or derivatives thereof, or hydrotyrosol, or dihydroxyphenol or their analogues, or derivatives thereof, or elenolic acid ortheir analogues, or derivatives thereof, or an iridoid glycoside, or asecoiridoid glycoside or their analogues, or derivatives thereof, or anycombinations of any of the foregoing thereof including but not limitedto olive leave extract, also modulate the expression of all of the threetypes of PPARs α, δ, and γ resulting in a coordinated regulation ofadipocyte differentiation, de-differentiation, transdifferentiation,adipocyte metabolism and energy homeostasis. In yet another embodiment,the lipogenic, lipolytic and energy uncoupling genes and gene productswhose expression is modulated includes, but is not limited to, PPAR δand its modulated genes and gene products. In yet another embodiment,the lipogenic, lipolytic and energy uncoupling genes whose expression ismodulated includes, but is not limited to, PPAR α and its modulatedgenes and gene products.

A second aspect of the invention provides a method of treating,controlling or preventing obesity, or of reducing body weight, or ofinhibiting fat accumulation, or of promoting fat burning and energyuncoupling in vivo, or of treating, controlling or preventing the onsetof one or more obesity-related disorders or conditions, comprisingadministering a therapeutically effective amount of oleuropein or ananalogue, or derivative thereof, or oleuropein aglycone or theiranalogues, or derivatives thereof, or hydrotyrosol, or dihydroxy phenolor their analogues, or derivatives thereof, or elenolic acid or theiranalogues, or derivatives thereof, or an iridoid glycoside, or asecoiridoid glycoside or their analogues, or derivatives, or anycombinations of any of the foregoing thereof including but not limitedto olive leave extract, to a mammalian subject in need thereof.

In one embodiment, the one or more obesity-related disorders orconditions are selected from the group consisting of coronary arterydisease, hypertension, stroke, peripheral vascular disease, insulinresistance, glucose intolerance, diabetes mellitus, hyperglycemia,hyperlipidemia, hypercholesteremia, hypertriglyceridemia,hyperinsulinemia, atherosclerosis, cellular proliferation andendothelial dysfunction, diabetic dyslipidemia, HIV-relatedlipodystrophy and metabolic syndrome, type II diabetes, diabeticcomplications including diabetic neuropathy, nephropathy, retinopathy orcataracts, heart failure, inflammation, thrombosis, congestive heartfailure, any other asthmatic or pulmonary disease related to obesity andany other viral infection infection related diseases and any othercardiovascular disease related to obesity or an overweight condition.

A third aspect provides a method of treating, controlling or preventingthe onset of one or more obesity related disorders or conditionsselected from the group consisting of Asthma and related diseasesincluding but not limited to, Allergy, Atopic Dermatitis (Eczema),Gastroesophageal Reflux Disease, Airway, Pulmonary and Lung disorders,in a mammalian subject in need of treatment, comprising administering atherapeutically effective amount of oleuropein or an analogue,derivative or oleuropein aglycone or their analogues, or derivativesthereof, or hydrotyrosol, or dihydroxy phenol or their analogues, orderivatives thereof, or elenolic acid or their analogues, or derivativesthereof, or an iridoid glycoside, or a secoiridoid glycoside or theiranalogues, or derivatives, or any component or any combinations of anyof the foregoing thereof including but not limited to olive leaveextract.

A fourth aspect provides a method of treating, controlling or preventingthe onset of one or more obesity related disorders or conditionsselected from the group consisting of AIDS and HAART related diseasesincluding but not limited to lipodystrophy in a human and or mammaliansubject in need of treatment, comprising administering a therapeuticallyeffective amount of oleuropein or an analogue, derivative or oleuropeinaglycone or their analogues, or derivatives thereof, or hydrotyrosol, ordihydroxy phenol or their analogues, or derivatives thereof, or elenolicacid or their analogues, or derivatives thereof, or an iridoidglycoside, or a secoiridoid glycoside or their analogues, orderivatives, or any component or any combinations of any of theforegoing thereof including but not limited to olive leave extract.

A fifth aspect provides a method of treating, controlling or preventingthe onset of one or more viral infection related disorders or conditionsselected from the group consisting of viral infection related diseasesincluding but not limited to the AIDS virus HIV-1, and other virusesincluding but not limited to simian immunodeficiency viruses (SIV),Sendai virus, feline immunodeficiency virus (FIV), respiratory syncytialvirus (RSV), measles virus, Ebola virus, Nipah and Hendra viruses, thesevere acute respiratory syndrome associated coronavirus (SARS-CoV), andthe avain flu virus in a humans, mammalian, avian, poultry, non humanprimates subjects in need of treatment, comprising administering atherapeutically effective amount of oleuropein or an analogue, orderivative, or oleuropein aglycone or their analogues, or derivativesthereof, or hydrotyrosol, or dihydroxy phenol or their analogues, orderivatives thereof, or elenolic acid or their analogues, or derivativesthereof, or an iridoid glycoside, or a secoiridoid glycoside or theiranalogues, or derivatives, or any component or any combinations of anyof the foregoing thereof including but not limited to olive leaveextract.

A sixth aspect of the invention provides a method for modulatingPeroxisome Proliferator Activation Receptor (PPAR) activity, comprisingadministering to a mammal in need thereof a therapeutically effectiveamount of oleuropein or an analogue, or derivative thereof, oroleuropein aglycone or their analogues, or derivatives thereof, orhydrotyrosol, or dihydroxy phenol or their analogues, or derivativesthereof, or elenolic acid or their analogues, or derivatives thereof, oran iridoid glycoside, or a secoiridoid glycoside or their analogues, orderivatives, or any component or any combinations of any of theforegoing thereof. or an iridoid glycoside, or a secoiridoid glycosideor their analogues, or derivatives, or any components or anycombinations thereof including but not limited to olive leave extract.

A seventh aspect provides a method of treating a subject suffering from,or at risk for developing a disease or condition for which PPARmodulation provides a therapeutic benefit, comprising administering tosaid subject a therapeutically effective amount of oleuropein, or ananalogue or derivative thereof or oleuropein aglycone or theiranalogues, or derivatives thereof, or hydrotyrosol, or dihydroxy phenolor their analogues, or derivatives thereof, or elenolic acid or theiranalogues, or derivatives thereof, or an iridoid glycoside, or asecoiridoid glycoside or their analogues, or derivatives, or anycomponent or any combinations of any of the foregoing thereof includingbut not limited to olive leave extract.

In one embodiment, the subject is a human or a non-human mammal. In apreferred embodiment, the subject is a mammal. In another preferredembodiment, the subject is a non-human mammal selected from the groupconsisting of cows, horses, pigs, sheep, goats, birds, rodents, dogs,cats and other domestic animals or farm animals.

In yet another particular embodiment, the condition for which PPARmodulation provides a therapeutic benefit is selected from the groupconsisting of obesity, obesity-related disorders and the sequelaethereof. In yet another embodiment, accordingly, the present inventionprovides for a method of treating inflammatory diseases or conditionscomprising administering a PPAR gamma modulator to a subject in need ofsuch therapy. In one particular embodiment, the method provides fortreating neurological or neurodegenerative diseases or conditions causedin part by the presence or influx of inflammatory cells, such as forexample, multiple sclerosis, stroke or Alzheimer's disease. The use of aPPAR modulator for treating a nervous system injury is alsocontemplated, for example, a spinal cord injury or traumatic braininjury. The use of a PPAR modulator for wound healing is alsocontemplated. In one particular embodiment, the PPAR modulator is a PPARgamma, alpha, or delta agonist, or a combined agonist.

In yet another particular embodiment, the PPAR is selected from PPAR δ,γ or α, or a dual, or pan PPAR δ, γ and α modulators.

In yet another particular embodiment, the obesity-related disorder orsequelae is selected from the group consisting of coronary arterydisease, hypertension, stroke, peripheral vascular disease, insulinresistance, diabetes mellitus, hyperlipidermia, atherosclerosis,cellular proliferation and endothelial dysfunction, diabeticdyslipidemia, type II diabetes, hyperinsulinemia, diabetic complicationsincluding diabetic neuropathy, nephropathy, retinopathy or cataracts,heart failure, hypercholesterolemia, inflammation, thrombosis,congestive heart failure, and any other cardiovascular disease relatedto obesity or an overweight condition.

An eighth aspect of the invention provides a method of reducing orpreventing formation of atherosclerotic lesions or preventingdiet-induced atherogenesis, comprising administering to a mammal in needthereof a therapeutically effective amount of oleuropein or an analogueor derivative thereof, or oleuropein aglycone or their analogues, orderivatives thereof, or hydrotyrosol, or dihydroxy phenol or theiranalogues, or derivatives thereof, or elenolic acid or their analogues,or derivatives thereof, or an iridoid glycoside, or a secoiridoidglycoside or their analogues, or derivatives, or any combinations of anyof any of the foregoing thereof.

A ninth aspect of the invention provides a method of modulatingendothelial dysfunction comprising administering to a mammal in needthereof a therapeutically effective amount of oleuropein or an analogue,or derivative, or oleuropein aglycone or their analogues, or derivativesthereof, or hydrotyrosol, or dihydroxy phenol or their analogues, orderivatives thereof, or elenolic acid or their analogues, or derivativesthereof, or an iridoid glycoside, or a secoiridoid glycoside or theiranalogues, derivatives, or any combinations of any of the foregoingthereof including but not limited to olive leave extract.

A tenth aspect of the invention provides a method for treating obesityor obesity related disorders or other disorders for which PPARmodulation provides a therapeutic benefit comprising administering asecond agent in combination with oleuropein, or an analogue orderivative thereof, or oleuropein aglycone or their analogues, orderivatives thereof, or hydrotyrosol, or dihydroxy phenol or theiranalogues, or derivatives thereof, or elenolic acid or their analogues,or derivatives thereof, or an iridoid glycoside, or a secoiridoidglycoside or their analogues, derivatives, or any combination of any ofthe foregoing thereof including but not limited to olive leave extract.

In one particular embodiment, the second agent is selected from thegroup consisting of a different PPAR modulating agent, a cholesterol orlipid lowering agent, a biguanide, insulin, an antihyperglycemic agent,GLP-1 or analogues thereof, DPP4 inhibitors, a weight loss agent, andany agent useful for treating metabolic syndrome or type 2 diabetes.

In another particular embodiment, the different PPAR modulating agent isa thiazolidinedione selected from the group consisting of troglitazone,pioglitazone and rosiglitazone.

In yet another particular embodiment, the cholesterol or lipid loweringagent is a HMG-CoA reductase inhibitor, and wherein the HMG-CoAreductase inhibitor is a statin selected from the group consisting ofatorvastatin, bervastatin, cerivastatin, dalvastatin, fluvastatin,itavastatin, lovastatin, mevastatin, nicostatin, nivastatin, pravastatinand simvastatin. In yet another particular embodiment, the cholesterolor lipid lowering agent is niacin or a fibrate.

In yet another particular embodiment, the biguanide is selected from thegroup consisting of metformin, phenformin, and buformin.

In yet another particular embodiment, the antihyperglycemic is aprandial glucose regulator or an alpha-glucosidase inhibitor.

In yet another particular embodiment, the prandial glucose regulator isrepaglinide or nateglinide.

In yet another particular embodiment, the alpha-glucosidase inhibitor isselected from the group consisting of acarbose, voglibose and miglitol.

In yet another particular embodiment, the agent useful for treatingmetabolic syndrome or type 2 diabetes is a sulfonylurea selected fromthe group consisting of glimepiride, glibenclamide (glyburide),gliclazide, glipizide, gliquidone, chloropropamide, tolbutamide,acetohexamide, glycopyramide, carbutamide, glibonuride, glisoxepid,glybuthiazole, glibuzole, glyhexamide, glymidine, glypinamide,phenbutamide, tolcylamide and tolazamide.

In yet another particular embodiment, the agent useful for treating AIDSand Highly Active Anti-Retroviral Therapy (HAART)-induced lipodystrophy,metabolic syndrome or type 2 diabetes is a thiazolidinedione (TZD) and afibrate.

In yet another particular embodiment, the agent useful for treatingHIV-1 infection and AIDS is a component or the full composition of theHighly Active Anti-Retroviral Therapy (HAART), including a proteaseinhibitor (PI), a nucleotide reverse transcriptase inhibitor (NRTI), anda non-nucleotide reverse transcriptase inhibitor (NNRTI).

In yet another particular embodiment, the agent useful for treatingobesity induced asthma and related disorders is selected from the groupconsisting of a glucocorticoid, an antileukotriene and an antihistamine.

An eleventh aspect of the invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of oleuropein,or an analogue or derivative thereof or oleuropein aglycone or theiranalogues, or derivatives thereof, or hydrotyrosol, or dihydroxy phenolor their analogues, or derivatives thereof, or elenolic acid or theiranalogues, or derivatives thereof, or an iridoid glycoside, or asecoiridoid glycoside or their analogues, derivatives, or anycombinations thereof including but not limited to olive leave extract,and a pharmaceutically acceptable carrier for delivery to a mammal inneed of such therapy.

In one particular embodiment, the pharmaceutical composition may beadministered orally, nasally, transdermally, intravenously,intramuscularly, or subcutaneously.

In another particular embodiment, the subject has or is at risk forunwanted weight gain, obesity or an obesity related disorder, e.g.,diabetes or glucose intolerance, insulin resistant states, hypertension,HIV-1 infection, or any of the other disorders disclosed herein. Inpreferred embodiments, the method includes identifying a subject asbeing in need of treatment or prevention of unwanted weight gain,obesity or an obesity related disorder. In another particularembodiment, the pharmaceutical composition is formulated for delivery toa human or non-human mammal. In a preferred embodiment, the mammal is ahuman. In another preferred embodiment, the subject is a non-humanmammal selected from the group consisting of cows, horses, pigs, sheep,goats, birds, rodents (including rats, mice and gerbils), dogs, cats andother domestic animals or farm animals.

A twelfth aspect of the invention provides a pharmaceutical compositiondirected to combination therapy, whereby oleuropein or oleuropeinaglycone or their analogues, or derivatives thereof, or hydrotyrosol, ordihydroxy phenol or their analogues, or derivatives thereof, or elenolicacid or their analogues, or derivatives thereof, or an iridoidglycoside, or a secoiridoid glycoside or their analogues or derivatives,or any combination thereof including but not limited to olive leaveextract, is combined with one or more other therapeutic agents that areuseful in the treatment of disorders associated with the development andprogression of obesity and obesity related disorders, such asatherosclerosis, hypertension, hyperlipidemias, dyslipidemias, diabetesand other related disorders as described herein.

In one particular embodiment, the composition for combination therapycomprises oleuropein and at least one other agent. In another particularembodiment, the composition for combination therapy comprises oleuropeinand at least two or more other agents.

In one particular embodiment, the at least one other therapeutic agentis a different PPAR modulating agent other than oleuropein.

In a more particular embodiment, the different PPAR modulating agent isa thiazolidinedione selected from the group consisting of troglitazone,pioglitazone and rosiglitazone.

In yet another particular embodiment, the at least one other therapeuticagent is a cholesterol-lowering agent.

In yet another particular embodiment, the cholesterol lowering agent isa HMG-CoA reductase inhibitor.

In yet another particular embodiment, the HMG-CoA reductase inhibitor isa statin selected from the group consisting of atorvastatin,bervastatin, cerivastatin, dalvastatin, fluvastatin, itavastatin,lovastatin, mevastatin, nicostatin, nivastatin, pravastatin andsimvastatin.

In yet another particular embodiment, oleuropein is combined with atherapy useful for the treatment of metabolic syndrome or type 2diabetes.

In yet another particular embodiment, the therapy useful for thetreatment of metabolic syndrome or type 2 diabetes and its associatedcomplications is selected from the group consisting of a biguanide drug,(including metformin, phenformin and buformin), insulin (syntheticinsulin analogues, amylin) and oral antihyperglycemics.

In yet another particular embodiment, oleuropein is combined with anoral antihyperglycemic agent, which is a prandial glucose regulator oran alpha-glucosidase inhibitor. In yet another particular embodiment,the prandial glucose regulator is repaglinide or nateglinide. In yetanother particular embodiment, the alpha-glucosidase inhibitor isacarbose, voglibose or miglitol.

In yet another particular embodiment, oleuropein is combined with asulfonylurea.

In yet another particular embodiment, the sulfonylurea is selected fromthe group consisting of glimepiride, glibenclamide (glyburide),gliclazide, glipizide, gliquidone, chloropropamide, tolbutamide,acetohexamide, glycopyramide, carbutamide, glibonuride, glisoxepid,glybuthiazole, glibuzole, glyhexamide, glymidine, glypinamide,phenbutamide, tolcylamide and tolazamide.

In one particular embodiment, the composition comprises a mixture ofoleuropein or oleuropein aglycone or their analogues, or derivativesthereof, or hydrotyrosol, or dihydroxy phenol or their analogues, orderivatives thereof, or elenolic acid or their analogues, or derivativesthereof, or an iridoid glycoside, or a secoiridoid glycoside or theiranalogues or derivatives, or any combination thereof including but notlimited to olive leave extract, and the at least one or two otheragents, wherein the at least one or two other agents may be administeredprior to, concurrent with, or subsequent to, oleuropein or oleuropeinaglycone or their analogues, or derivatives thereof, or hydrotyrosol, ordihydroxy phenol or their analogues, or derivatives thereof, or elenolicacid or their analogues, or derivatives thereof, or an iridoidglycoside, or a secoiridoid glycoside or their analogues or derivatives,or any combination thereof including but not limited to olive leaveextract.

In another particular embodiment, the composition comprising oleuropeinor an analogue or derivative thereof or oleuropein aglycone or theiranalogues, or derivatives thereof, or hydrotyrosol, or dihydroxy phenolor their analogues, or derivatives thereof, or elenolic acid or theiranalogues, or derivatives thereof, or an iridoid glycoside, or asecoiridoid glycoside or their analogues or derivatives, or anycombination thereof including but not limited to olive leave extract,interacts with, e.g., binds to, PPARδ, PPARγ and PPARα.

In yet another particular embodiment, oleuropein or oleuropein aglyconeor their analogues, or derivatives thereof, or hydrotyrosol, ordihydroxy phenol or their analogues, or derivatives thereof, or elenolicacid or their analogues, or derivatives thereof, or an iridoidglycoside, or a secoiridoid glycoside or their analogues or derivatives,or any combination thereof including but not limited to olive leaveextract, is combined with a therapy useful for the treatment of the AIDSvirus HIV-1, and other viruses with a type I transmembrane envelopeglycoprotein, such as simian immunodeficiency viruses (SIV), Sendaivirus, feline immunodeficiency virus (FIV), respiratory syncytial virus(RSV), measles virus, Ebola virus, Nipah and Hendra viruses, the severeacute respiratory syndrome associated coronavirus (SARS-CoV), and theavain flu virus H5N1 including but not limited to PI, NRTI, NNRTI,HAART, tamiflu, ribavirin, steroid, recombinant nematode anticoagulantprotein c2 (rNAPC2).

In yet another particular embodiment, oleuropein is combined with atherapy useful for the treatment of obesity induced asthma and relateddisorders, including but not limited to the PPARs, glucocorticoids,antileukotrienes and antihistamines.

In another particular embodiment, the oleuropein or an analogue orderivative thereof, or oleuropein aglycone or their analogues, orderivatives thereof, or hydrotyrosol, or dihydroxy phenol or theiranalogues, or derivatives thereof, or elenolic acid or their analogues,or derivatives thereof, an iridoid glycoside, or a secoiridoidglycoside, or their analogues, or derivatives thereof, or anycombination thereof including but not limited to olive leave extract, istargeted to adipose tissue in a subject. The oleuropein or an analogueor derivative thereof or oleuropein aglycone or their analogues, orderivatives thereof, or hydrotyrosol, or dihydroxy phenol or theiranalogues, or derivatives thereof, or elenolic acid or their analogues,or derivatives thereof, or an iridoid glycoside, or a secoiridoidglycoside or an analogue, or derivative thereof, or any combinationthereof including but not limited to olive leave extract, may betargeted to adipose tissue by virtue of an inherent characteristic,e.g., lipid solubility. In other embodiments, the agent may include(e.g., the agent can be linked, fused or conjugated to, or enveloped in)a targeting reagent that targets the agent to an adipose tissue. Thetargeting reagent can be a nucleic acid, a protein (e.g., a hormone,e.g., leptin, conjugate or an antibody to an adipocyte-specificantigen), a lipid (e.g., a liposome), a carbohydrate, or other moleculethat is targeted to an adipose tissue.

In yet another particular embodiment, the oleuropein or an analogue orderivative thereof, or oleuropein aglycone or their analogues, orderivatives thereof, or hydrotyrosol, or dihydroxy phenol or theiranalogues, or derivatives thereof, or elenolic acid or their analogues,or derivatives thereof, or an iridoid glycoside, or a secoiridoidglycoside, or their analogues or derivatives thereof, or any combinationof the foregoing including but not limited to olive leave extract, istargeted to adipose tissue via Resistin (resistance to insulin) orResistin-Like Molecules (RELMs or FIZZ1-3) (Degawa-Yamauchi M, SerumResistin (FIZZ3) Protein Is Increased in Obese Humans, J. Clin.Endocrinol. Metab., 88: 5452-5455, (2003); (Diabetologia. (2006) May 10;Anal Chem. (2006) May 15; 78(10):3271-6; Hum Reprod. (2006) May 12) orAdipocyte-Specific Secretory Factor (ADSF) Proteins (Endocrine. (2006)February; 29(1):81-90 Proc Natl Acad Sci USA. (2004) April 27;101(17):6780-5. Epub (2004) April 16) or antibodies to such proteins, aswell as Leptin (Int J Obes (Lond). (2006) May 16) or Leptin Receptorantibodies, Acrp30/Adiponectin (Endocrinology. (2006) June;147(6):2690-5. Epub (2006) March 2; (Hum Reprod. (2006) May 12; [Epubahead of print]; Biochem Biophys Res Commun. (2006) June 23;345(1):332-9. Epub (2006) April 27. J Endocrinol Invest. (2006) March;29(3):231-6;) (Hum Reprod. 2006 May 12; [Epub ahead of print]; BiochemBiophys Res Commun. 2006 Jun. 23; 345(1):332-9. Epub 2006 Apr. 27. JEndocrinol Invest. 2006 March; 29(3):231-6;) or Adipsin antibodies,Orexins (Br J Nutr. 2004 August; 92 Suppl 1:S47-57) or Orexins Receptorantibodies, a Glucose Transporter (Glut1-Glut14) (Clin Exp PharmacolPhysiol. 2006 April; 33(4):395-9; Am J Med. 2006 May; 119(5 Suppl1):S10-6) antibody, or an antibody to a Hypoxia Induced Factor(HIF-alpha, beta) (HIF-alpha, beta, Diabetologia. 2006 May;49(5):1049-63. Epub 2006 Feb. 28; Biochem Biophys Res Commun. 2006 Mar.10; 341(2):549-56.) Am J Physiol Endocrinol Metab. 2006 March;290(3):E591-7. Epub 2005 Oct. 18.

In yet another particular embodiment, the oleuropein or an analogue orderivative thereof, or oleuropein aglycone or their analogues, orderivatives thereof, or hydrotyrosol, or dihydroxy phenol or theiranalogues, or derivatives thereof, or elenolic acid or their analogues,or derivatives thereof, or an iridoid glycoside, or a secoiridoidglycoside or their analogue or derivative, or any combination thereofincluding but not limited to olive leave extract, is targeted to apreadipocyte and its specific proteins such as pref-1 and C1q tomodulate adipocyte differentiation and maturation.

In yet another particular embodiment, the oleuropein or an analogue orderivative thereof, or oleuropein aglycone or their analogues, orderivatives thereof, or hydrotyrosol, or dihydroxy phenol or theiranalogues, or derivatives thereof, or elenolic acid or their analogues,or derivatives thereof, or an iridoid glycoside, or a secoiridoidglycoside, or their analogues, or derivatives thereof, or anycombination of the foregoing including but not limited to olive leaveextract, is targeted to a mesenchymal stem cell (MSC) or an embryonicstem cell. MSCs differentiate into adipocytes, chondrocytes,osteoblasts, and myoblasts. Thus, these stem cells are promisingcandidates for adipogenesis management by oleuropein and derivatives oroleuropein aglycone or their analogues, or derivatives thereof, orhydrotyrosol, or dihydroxy phenol or their analogues, or derivativesthereof, or elenolic acid or their analogues, or derivatives thereof, oran iridoid glycoside, or a secoiridoid glycoside or their analogue orderivative, or any combination thereof including but not limited toolive leave extract, targeting and/or ex-vivo treatment so as tomodulate adipocyte differentiation, de-differentiation andtrans-differentiation. The application of oleuropein and derivatives inthis system is not only effective in treating obesity and relatedmetabolic syndromes by modulating adipocyte differentiation anddevelopment but also provide tools to manipulate adult stem cells forcell-based approaches in regenerative medicine. For example, aged andosteoporotic patients have a high fat to bone ratio in their bonescompared with young and healthy counterparts, an outcome possibly due tothe conversion of bone to fat cells. Application of oleuropein or ananalogue or derivative thereof, or oleuropein aglycone or theiranalogues, or derivatives thereof, or hydrotyrosol, or dihydroxy phenolor their analogues, or derivatives thereof, or elenolic acid or theiranalogues, or derivatives thereof, or an iridoid glycoside, or asecoiridoid glycoside or their analogue or derivative, or anycombination thereof including but not limited to olive leave extract tomediate transdifferentiation between osteoblasts and adipocytes shouldbe of relevance to the development of therapeutic control of bone lossin osteoporosis. For specific targeting of MSC, these cells expressleukemia inhibitory factor, macrophage colony-stimulating factor, andstem cell factor specifically. Thus, these proteins and antibodiesspecific for or that bind these proteins can be used for targeting.

In another particular embodiment, the targeting reagent is lipidsoluble.

In another particular embodiment, the administration of the compositionsof the invention can be initiated, e.g., (a) when the subject begins toshow signs of unwanted weight gain, obesity or an obesity-relateddisease; (b) when obesity or an obesity-related disease is diagnosed;(c) before, during or after a treatment for obesity or anobesity-related disease is begun or begins to exert its effects; or (d)generally, as is needed to maintain health, e.g., normal weight. Theperiod over which the agent is administered (or the period over whichclinically effective levels are maintained in the subject) can be longterm, e.g., for six months or more or a year or more, or short term,e.g., for less than a year, six months, one month, two weeks or less.

In another particular embodiment, the pharmaceutical compositionsdescribed herein, including oleuropein, or an analogue or derivativethereof, or oleuropein aglycone or their analogues, or derivativesthereof, or hydrotyrosol, or dihydroxy phenol or their analogues, orderivatives thereof, or elenolic acid or their analogues, or derivativesthereof, or an iridoid glycoside, or a secoiridoid glycoside or theiranalogues, or derivatives thereof, or any combination thereof includingbut not limited to olive leave extract and any second agent to beadministered with oleuropein or its analogues or derivatives thereof, oroleuropein aglycone or their analogues, or derivatives thereof, orhydrotyrosol, or dihydroxy phenol or their analogues, or derivativesthereof, or elenolic acid or their analogues, or derivatives thereof, oran iridoid glycoside, or a secoiridoid glycoside or their analogues orderivatives thereof, or any combination thereof including but notlimited to olive leave extract as described above, are administered in atherapeutically effective dose.

In yet another particular embodiment, oleuropein or an analogue, orderivative thereof, or oleuropein aglycone or their analogues, orderivatives thereof, or hydrotyrosol, or dihydroxy phenol or theiranalogues, or derivatives thereof, or elenolic acid or their analogues,or derivatives thereof, or an iridoid glycoside, or a secoiridoidglycoside, or their analogues, or derivatives thereof, or anycombination thereof including but not limited to olive leave extract mayhave an effect on white or brown adipose tissue or a combinationthereof. For example, since PPAR delta is involved in thermogenesis andenergy uncoupling (the main function of brown fat) and oleuropein hashigh affinity for PPAR delta, it may also have an effect on brownadipose tissue. Accordingly, oleuropein may be involved in thedissipation of stored fat as heat in brown adipose tissue. Brown fatplays an important role in the control of body weight, and mitochondrialuncoupling proteins may be one of many factors involved in thedevelopment of obesity. An interesting demonstration of this is found ina report in which transgenic mice with genetic ablation of brown fatdeveloped obesity in the absense of overeating (Bachman E S, Dhillon H,Zhang C—Y, et al. BetaAR signaling required for diet-inducedthermogenesis and obesity resistance. Science 297:843, 2002).

A thirteenth aspect of the invention provides a method for identifying acandidate compound or an analogue or derivative of oleuropein thatmodulates adipocyte differentiation, de-differentiation,trans-differentiation, fat accumulation and adipogenic gene expressionor for treating obesity or obesity-related disorders, comprising:

-   -   a. treating a cell with an inducing agent in the presence or        absence of a candidate compound;    -   b. determining whether the candidate compound inhibits        differentiation of the pre-adipocyte cell to an adipocyte, or        whether the candidate compound down-regulates adipogenic and        lipogenic gene or gene product expression and/or up-regulates        lipolytic and/or an energy uncoupling gene or gene product        expression;    -   c. comparing the results obtained with the candidate compound in        vitro with the results obtained using oleuropein;    -   d. testing the candidate compound and oleuropein in an animal        model of obesity to determine the in vivo effects of both the        candidate compound and oleuropein;    -   e. determining whether the candidate compound decreases the        amount of adipose tissue in vivo or whether the test compound        prevents further fat accumulation in vivo, and    -   f. selecting a candidate compound that has equivalent or better        activity than oleuropein.

In one particular embodiment, the invention relates to a method foridentifying a therapeutic agent having analogous activity to oleuropeincomprising treating a type of cell that expresses a type of PPAR with acandidate compound/therapeutic; and determining the level of expressionof at least one gene selected from the group consisting of PPARγ2,lipoprotein lipase (LPL), and αP2 lipid binding protein, wherein achange in the profile of expression of at least one of these genes inthe cell treated with the candidate compound/therapeutic relative to acell that was not treated with the candidate compound/therapeuticindicates that the candidate compound/therapeutic is a therapeutic fortreating a disease associated with a PPAR. The “profile”, “profiling” or“profile of expression” refers to both the level of expression of one ormore genes and also the activity or function of one or more of thesegenes. Accordingly, the candidate compound may have an effect not onlyon the expression of one or more genes, but on the function or activityof one or more genes. In one particular embodiment, an increase in theprofile of expression of at least one of these genes following treatmentwith a candidate compound/therapeutic indicates that the candidatecompound/therapeutic is a therapeutic for treating a disease associatedwith a PPAR. The profile in expression of one or more of these genesrelates to the differentiation or maturation of the adipocyte. Inanother particular embodiment, the inducing agent that is used for invitro analysis is any compound or molecule that induces differentiationof a pre-adipocyte or a mesenchymal stem cell into a mature adipocyte,eg. dexamethasone, insulin and methyl xanthine. In another particularembodiment, following contacting/treating a cell containing PPAR with acandidate compound/therapeutic, one may determine the profile ofexpression of other genes selected from the group consisting of leptin,TNFα, IL-6, PAI-1, adipsin, complement factor C3 and angiotensinogen. Inyet another particular embodiment, in addition to classic markers ofadipogenesis such as hormone-sensitive lipase (HSL), lipoprotein lipase(LPL), adiponectin, fatty acid-binding protein 4, perilipin and CCAATenhancer binding protein, and enzymes of energy metabolism such asglycerol-3-phosphate dehydrogenase 1, acetyl-coenzyme A carboxykinase,phosphoenolpyruvate carboxykinase and pyruvate dehydrogenase kinase,profiles on the expression of other related genes including PPARα,PPARδ, PPARγ, AP2, MARPK3, SREBP-1, leptin, and GLUT4 are envisionedfollowing treatment of a type of cell with a candidatecompound/therapeutic. These are important because they are not onlyadipocyte “markers” but also important in adipocyte function andpathogenesis. Any agent that affects adipogenesis would modulate theexpression of these genes. In one particular embodiment, an adipogenicagent would up-regulate while an anti-adipogenic agent would downregulate the expression of one or more of these genes. However,physiologically it is not a simple up or down regulation. It involvesthe regulation of other related genes upstream and downstream asincluding PPARδ, PPARα, enzymes involve in lipid metabolism, fatty acidoxidation, energy uncoupling as well as genes modulated by the PPARs. Inaddition to monitoring the up or down regulation of these genes, anotherparticular embodiment provides for measuring the differential expressionas well as polymorphisms of these genes. In yet another particularembodiment, the PPAR is selected from the group consisting of α, γ, orγ.

In another embodiment of the invention, the candidate therapeutic isselected from the group consisting of proteins, peptides,peptidomimetics, antibodies, nucleic acids, including RNA (eg. siRNA),DNA, derivatives of fatty acids, and small molecules. The smallmolecules may be synthetic or may be derived from a natural source, suchas a plant, animal, microbe, or soil.

In another embodiment, the disease is obesity or an obesity-relateddisorder, such as, but not limited to, Type II diabetes.

In another embodiment, the expression level of at least one of the genesnoted above is detected. In another particular embodiment, theexpression level of at least two or more of the genes noted above isdetected.

In another embodiment, the composition is an oral capsule or tablet, aliquid suspension, or a chip or wafer for oral delivery. In anotherembodiment, the composition is formulated for intravenous use, forintramuscular use, for subcutaneous delivery or for intraperitonealinjection.

In another embodiment, the composition comprising oleuropein or ananalogue, derivative or oleuropein aglycone or their analogues, orderivatives thereof, or hydrotyrosol, or dihydroxy phenol or theiranalogues, or derivatives thereof, or elenolic acid or their analogues,or derivatives thereof, or an iridoid glycoside, or a secoiridoidglycoside, or their analogues, or derivatives thereof, or anycombination thereof including but not limited to olive leave extract maybe targeted to (e.g., the oleuropein or analogue or derivative thereofcan be linked, fused or conjugated to, or enveloped in) the adipocyte byattachment of the oleuropein or analogue or derivative thereof toadipose tissue via Resistin or Resistin-Like Molecules (RELMs orFIZZ1-3) or Adipocyte-Specific Secretory Factor (ADSF) Proteins orantibodies to such proteins, as well as Leptin or Leptin Receptorantibodies, Acrp30/Adiponectin or Adipsin antibodies, Orexins or OrexinsReceptor antibodies, a Glucose Transporter (Glut1-Glut14) antibody, oran antibody to a Hypoxia Induced Factor (HIF-alpha, beta).

In yet another particular embodiment, the composition comprising theoleuropein or an analogue or derivative thereof, or oleuropein aglyconeor their analogues, or derivatives thereof, or hydrotyrosol, ordihydroxy phenol or their analogues, or derivatives thereof, or elenolicacid or their analogues, or derivatives thereof, or an iridoidglycoside, or a secoiridoid glycoside, or their analogues, orderivatives thereof, or any combination thereof including but notlimited to olive leave extract, is targeted to a preadipocyte and itsspecific proteins such as Preadipocyte factor-1 (Pref-1), which is anepidermal growth factor-like domain-containing transmembrane protein andis implicated in inhibiting preadipocytes differentiation and componentof complement (C1) or C1q, which is a serum glycoprotein involved inimmune complexes to modulate adipocyte differentiation and maturation.

In yet another particular embodiment, the composition comprising theoleuropein or an analogue or derivative thereof, or oleuropein aglyconeor their analogues, or derivatives thereof, or hydrotyrosol, ordihydroxy phenol or their analogues, or derivatives thereof, or elenolicacid or their analogues, or derivatives thereof, or an iridoidglycoside, or a secoiridoid glycoside, or their analogues, orderivatives thereof, or any combination thereof including but notlimited to olive leave extract, is targeted to a mesenchymal stem cell(MSC). MSCs differentiate into adipocytes, chondrocytes, osteoblasts,and myoblasts. Accordingly, in yet another embodiment, the inventionprovides a method of transdifferentiating adipocytes into osteoblasts,myoblasts and chondrocytes, wherein the method comprises administeringto a mammal in need thereof a therapeutically effective amount ofoleuropein or an analogue, or derivative thereof, or oleuropein aglyconeor their analogues, or derivatives thereof, or hydrotyrosol, ordihydroxy phenol or their analogues, or derivatives thereof, or elenolicacid or their analogues, or derivatives thereof, or an iridoidglycoside, or a secoiridoid glycoside, or their analogues, orderivatives thereof, or any combination thereof including but notlimited to olive leave extract. Thus, these stem cells are promisingcandidates for adipogenesis management by oleuropein and derivatives (asmentioned above) targeting and/or ex-vivo treatment so as to modulateadipocyte differentiation, trans-differentiation and de-differentiation.The application of oleuropein and derivatives (as mentioned above) inthis system is not only effective in treating obesity and relatedmetabolic syndromes by modulating adipocyte differentiation anddevelopment but also provide tools to manipulate adult stem cells forcell-based approaches in regenerative medicine. For example, aged andosteoporotic patients have a high fat to bone ratio in their bonescompared with young and healthy counterparts, an outcome possibly due tothe conversion of bone to fat cells. Application of oleuropein or ananalogue, or derivative thereof, or oleuropein aglycone or theiranalogues, or derivatives thereof, or hydrotyrosol, or dihydroxy phenolor their analogues, or derivatives thereof, or elenolic acid or theiranalogues, or derivatives thereof, or an iridoid glycoside, or asecoiridoid glycoside, or their analogues, or derivatives thereof, orany combination thereof including but not limited to olive leave extractto mediate transdifferentiation between osteoblasts and adipocytesshould be of relevance to the development of therapeutic control of boneloss in osteoporosis. For specific targeting of MSC, these cells expressleukaemia inhibitory factor, macrophage colony-stimulating factor, andstem cell factor specifically. Thus these proteins and their antibodiescan be used for targeting.

In another particular embodiment, the targeting reagent is lipidsoluble.

In a fourteenth aspect, the invention provides a method of determiningwhether a subject is responsive to treatment with a therapeutic such asoleuropein or a therapeutic having analogous activity to oleuropein,comprising determining the level of expression of one or more genes orgene products selected from the group consisting of PPARγ2, LPL, or αP2in a cell or a bodily fluid sample of the subject, and/or determiningthe differential expression of the adipogenic, lipogenic and lipolyticgenes or gene products, wherein a change in level of expression of anyone or more of these genes or gene products in a cell or bodily fluidsample of the subject relative to that in a cell or bodily fluid sampleof a subject that was not treated with oleuropein or a therapeutichaving analogous activity to oleuropein, or a change of the profiles andor functions of adipogenic, lipogenic and lipolytic genes, indicatesthat the subject is responsive to treatment with oleuropein or anoleuropein analogue or derivative.

A fifteenth aspect of the invention provides a method for determiningwhether a subject is responsive to treatment with a therapeutic havinganalogous activity to oleuropein, comprising determining the profile ofexpression of adipogenic, lipogeneic and lipolytic, genes or geneproducts selected from the group consisting of PPARδ, PPARγ and PPARα incells of the subject, wherein a higher or lower level of expression orfunction/activity of any one of these genes in the cells of the subjectrelative to that in cells of a subject that was not treated with a PPARligand indicates that the subject is responsive to treatment with thePPAR ligand.

In one particular embodiment, the cells are obtained from whole blood,e.g. peripheral blood mononuclear cells (PBMC). In another particularembodiment, the cells are adipocytes. In yet another particularembodiment, the cells are pre-adipocytes. In yet another embodiment, thecells are mesenchymal stem cells. In yet another particular embodiment,the bodily fluid sample is plasma or serum.

In a sixteenth aspect, the invention provides a method for predictingwhether a subject would be responsive to treatment with a compoundhaving analogous activity to oleuropein, comprising treating the subjectwith the candidate or test compound followed by collecting preadipocytesor whole blood of the subject and determining the profile of expressionof at least one of the genes selected from the group consisting ofPPARγ2, LPL, αP2, leptin, TNFα, IL-6, PAI-1, adipsin, complement factorC3, angiotensinogen, hormone-sensitive lipase (HSL), lipoprotein lipase(LPL), adiponectin, fatty acid-binding protein 4, perilipin and CCAATenhancer binding protein, and enzymes of energy metabolism such asglycerol-3-phosphate dehydrogenase 1, acetyl-coenzyme A carboxykinase,phosphoenolpyruvate carboxykinase and pyruvate dehydrogenase kinase,wherein a change in the profile of expression (the level of expressionand/or the change in function or activity) of at least one of thesegenes relative to expression in cells or in a whole blood sample ofsubjects not treated with oleuropein, indicates that the subject wouldbe responsive to treatment with the compound having analogous activityto oleuropein. In another particular embodiment, a method for predictingwhether a subject would be responsive to treatment with a compoundhaving analogous activity to oleuropein, comprises incubating cells ofthe subject with oleuropein and determining the level of expression ofat least one of the genes selected from the group consisting of PPARαand PPARδ as well as their responsive genes in lipid metabolism, lipidutilization and energy uncoupling. The expression of these genes can beprofiled in a cellular sample or in a blood sample, for example, wholeblood, or plasma or serum. In addition to PPARα, PPARδ and PPARγ andtheir responsive genes, the levels of glucose, fasting insulin, insulinAUC, total cholesterol, LDL cholesterol, HDL cholesterol, triglyceride,adiponectin, free fatty acid and TNFα, are also indications of an effectof oleuropein or an analogue or derivative thereof.

In one embodiment, the PPAR is selected from the group consisting of α,γ, or γ.

A seventeenth aspect of the invention provides a method for purifyingbiologically active oleuropein, an oleuropein derivative, or anoleuropein metabolite from olive leave extract, the method comprisingthe steps:

-   -   a) extracting the oleuropein, or a derivative or metabolite        thereof by heating olive leaves to at least 80° C. but not above        85° C. for about 10-12 hours in water, saline or phosphate        buffer;    -   b) collecting the liquid from step a) and repeating the        extracting procedure of step a) at least one additional time;    -   c) combining the liquid from steps a) and b) and centrifuging at        least 20,000×g for at least 30 minutes to remove small        particulates or insoluble material;    -   d) concentrating the liquid from step c) by lyophilization until        dried;    -   e) dissolving the dried extract from step d) into sterile water        to a concentration of about 10-20 mg/ml and filter sterilizing;    -   f) distributing the material from step e) into sterile cryotubes        under aseptic conditions and storing at a temperature of at        least −80° C.;    -   g) fractionation, characterization and analyzing the extract by        a method selected from the group consisting of high pressure        liquid chromatography (HPLC), thin layer chromatography (TLC)        and liquid chromatography-mass spectrometry (LC-MS); and    -   h) comparing the material obtained in step g) with a known        standard.

In one embodiment, the ratio of dried leaves to water, saline orphosphate buffer in step c) above is about 1 gram of dried leaves toabout 40 ml of water, saline or phosphate buffer.

In another embodiment, the filter sterilizing is accomplished using afilter of about 0.45 microns.

An eighteenth aspect of the invention provides a method for synthesizingbiologically active hydroxytyrosol, or a hydroxytyrosol analogue orderivative, comprising:

-   -   a) providing 3,4-dihydroxylphenylacetic acid;    -   b) reacting 3,4-dihydroxylphenylacetic acid with acetyl chloride        and methanol by stirring at room temperature overnight to yield        3,4-dihydroxylphenylacetic ester    -   c) purifying the 3,4-dihydroxylphenylacetic ester by column        chromatography.    -   d) dissolving the 3,4-dihydroxylphenylacetic ester of step c) in        tetrahydrofuran;    -   e) adding 1 molar LiAlH4 into the reaction mixture from step d);    -   stirring at 0° C. for about 2 hours;    -   f) purifying the hydroxytyrosol, or a hydroxytyrosol analogue or        derivative by column chromatography; and    -   g) characterizing the hydroxytyrosol, or a hydroxytyrosol        analogue or derivative by liquid chromatography-mass        spectrometry.

A nineteenth aspect of the invention provides for the preparation ofbiologically active hydroxytyrosol, comprising:

-   -   a) providing oleuropein;    -   b) treating the oleuropein of step a) with beta-glycosidase to        yield oleuropein aglycone;    -   c) hydrolyzing the oleuropein aglycone to yield hydroxytyrosol        and elenolic acid.

In one embodiment, the hydrolyzing of step c) above is accomplished bytreating the oleuropein aglycone of step b) with an esterase to yieldhydroxytyrosol and elenolic acid.

A twentieth aspect of the invention provides for a method of inhibitinghuman immunodeficiency virus (HIV) infectivity, comprising administeringa therapeutically effective amount of oleuropein or hydroxytyrosol, or aderivative or analogue thereof. In one embodiment, the administering maybe in vitro or in vivo.

In another embodiment, the oleuropein or hydroxytyrosol, or a derivativeor analogue thereof, prevents the fusion of the virus to the host cell,and/or prevents cell to cell transmission of the virus, and/or preventsviral replication by binding to the active/catalytic site of the HIVintegrase.

In yet another embodiment, the inhibiting of HIV infectivity is theresult of the binding of oleuropein or hydroxytyrosol, or a derivativeor analogue thereof, to a conserved hydrophobic pocket on the surface ofthe central trimeric coiled-coil of the HIV gp41 fusion domain.

In yet another embodiment, the oleuropein or hydroxytyrosol, or aderivative or analogue thereof, interacts with the N-terminal heptadrepeat (NHR) coiled-coil trimer N36 helices and interferes with theformation of 6HB with the C-terminal heptad repeat (CHR), C34.

In yet another embodiment, the oleuropein or hydroxytyrosol, or aderivative or analogue thereof, inhibits 6HB formation.

In yet another embodiment, the oleuropein or hydroxytyrosol, or aderivative or analogue thereof, binds to both regions I and II of saidintegrase.

In yet another embodiment, the oleuropein or hydroxytyrosol, or aderivative or analogue thereof, inhibits one or more of the followingactivities of the integrase:

-   -   a) inhibition of 3′ processing activity of the HIV integrase;    -   b) inhibition of strand-transfer activity of the HIV integrase;        or    -   c) inhibition of the disintegration activity of the HIV        integrase.

In yet another embodiment, the human immunodeficiency virus is HIV-1 orHIV-2.

In yet another embodiment, the oleuropein or hydroxytyrosol, or aderivative or analogue thereof, inhibits the fusion or replication ofany one of an M tropic or a T tropic strain from different HIV clades.

Other objects and advantages will become apparent from a review of theensuing detailed description and attendant claims taken in conjunctionwith the following illustrative drawings. All references cited in thepresent application are incorporated herein in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Demonstrates that oleuropein modulates adipocyte differentiation

FIG. 2 Demonstrates that oleuropein de-differentiates adipocytes andallows transdifferentiation

FIG. 3 Represents aortas from apoE knock out mice fed a Western diet for4 months, and then stained with Oil Red 0

FIG. 4 Shows aortic lesions in apoE knock out mice fed a Western dietfor 4 months

FIG. 5 Shows the predicted PPARδ-oleuropein binding structure

FIG. 6 Shows the predicted PPARγ-oleuropein binding structure

FIG. 7 Shows the predicted PPARα-oleuropein binding structure

FIG. 8 Shows the chemical structure of oleuropein

FIG. 9 Shows the HPLC elution profile of Olive Leaf Extract (OLE). Thenumbers represent the identified peaks. The identities of the peaks areshown in Table 5. Peak 6 is oleuropein, and contains the bulk of thebiological activity.

FIG. 10 Shows the chemical structure of oleuropein and its majormetabolites.

FIG. 11 Shows the results of the standardization of olive leaf extractby liquid chromatography-mass spectrometry (LC-MS) based on oleuropeincontent.

FIG. 12 Shows the results of the quantitation of oleuropein andmetabolites in serum and urine of mice fed oleuropein.

FIG. 13. LC-MS analysis of Ole and HT, metabolism of Ole and chemicalsynthesis of HT

A. LC elution profile of Ole. B. MS analysis of Ole, showing one majorcomponent with a molecular mass of 539. C. LC elution profile of HT. D.MS analysis of HT, showing one major component with a molecular mass of153. E. Metabolism of Ole, showing major reactions in the production ofHT from Ole. F. Chemical synthesis of HT.

FIG. 14. HIV-1 gp41 and the formation of fusion core 6HB.

A. Structure map of HIV-1 gp41, FP (fusion peptide), NHR (N-terminalheptad repeat), CHR(C-terminal heptad repeat), TR (tryptophan-rich), TM(transmembrane), and CP (cytoplasmic) domains. Residue numberscorrespond to positions in gp160 of HIV-1HXB2. B. Formation of 6HB byN36 and C34 helices, showing the trimeric coiled-coil core of N36 andthe surrounding three C34s. C. and D. Effect of Ole and HT on fusioncore, 6HB formation, showing the binding of Ole or HT to the N36trimeric coiled-coil core thus inhibiting 6HB formation.

FIG. 15. Molecular docking of Ole and HT with HIV-1 gp41

A. Structures of the 5-helical gp41 bundle. B. Structure of Ole. The 9freely rotating bonds are shown. C. and D. The predicted bindingstructures of Ole (C) and HT (D) inside the HIV-1 gp41 hydrophobic site.gp41 is shown as a surface model and Ole and HT are shown as stickmodels. Both molecules for m stable hydrogen bonds with Q577 (green) onN36 peptide. E. and F. Ribbon representation showing hydrogen binding ofgp41 5HB with Ole (E) and HT (F). The trimeric coiled-coil core of N36peptides are pink and C34 peptides green. The Ole and HT molecules areshown as stick formation and hydrogen bonds with Q577 are in greendashed lines.

FIG. 16. The effect of Ole and HT on the formation of 6HB between HIV-1peptides N36 and C34

A. Native PAGE was carried out in Tris-glycine 18% gels, at 120 Vconstant voltages at room temperature for 2 h. The gel was then stainedwith Coomassie Blue and analyzed by densitometry. Lane 1, molecularweight markers; lane 2, N36; lane 3, C34; lane 4 fusion complex,(N36+C34); lanes 5-8 and 9-12, in the presence of Ole and HT at 25, 50,75 and 100 nM. B. CD analysis, CD spectra for N36 (Green), C34 (Red),(N36+C34) 6HB (Blue), and N36+C34 in the presence of 25 and 50 nM of Oleor HT.

FIG. 17. Molecular docking of Ole and HT with HIV-1 integrase

The predicted binding structures of Ole (A) and HT (B) inside the HIV-1integrase catalytic site. Integrase is shown as a surface model, whileOle and HT are shown as van der Waals models and the purple sphererepresents Mg²⁺. Hydrogen bonds formed by Ole (C) and HT (D) withintegrase are indicated as green dotted lines, and the integrasebackbone is represented by the cyan ribbon.

FIG. 18. The effect of Ole and HT on HIV-1 integrase 3′-processing,strand transfer and disintegration activities

A. The Effect of Ole and HT on 141V-1 Integrase 3′-Processing Activity

Left: Schematic representation of the 3′-processing activity of HIV-1integrase. A 5′-³²P-labeled 21-mer of HIV-1 LTR U3 double-stranded (ds)DNA was used as the substrate. Specific cleavage of the dinucleotide GTfrom the 3′ end of the substrate results in the formation of 19-mer3′-recessed U3-GT.Right: Inhibition of HIV-1 integrase 3′-processing activity by Ole andHT. Inhibition was monitored by the formation of labeled 19-mer product.Lane 1, the 21-mer substrate, 5′-³²P-labeled U3. Lane 2, cleavage of the3′ GT of the 21-mer substrate by HIV-1 integrase results in theformation of the 19-mer 3′-recessed U3-GT. Lanes 3-7 or 8-12, in thepresence of 25, 50, 75, 100, and 200 nM Ole or HT.

B. The Effect of Ole and HT on HIV-1 Integrase Strand Transfer Activity

Left: Schematic representation of strand transfer (integration).Pre-cleaved 5′-³²P-labeled U3-GT 19-mer was used as the viral substrateand, unlabeled pUC18 DNA (2.69 kb) was used as the heterologous targetsubstrate.Right: Inhibition of strand-transfer activity of HIV-1 integrase by Oleand HT. Integration was monitored by the conversion of the unlabeledplasmid into labeled DNA. Lanes 1,5′-³²P-labeled size marker, HindIIIfragments of λ phage DNA. Lanes 2, target substrate, pUC18; because itis unlabeled, it is not seen in the autoradiogram. Lanes 3, the productof integration (ST) by HIV-1 integrase. The integration of the5′³²P-labeled U3-GT into pUC18 results in the appearance of labeled bandat 2.69 kb, corresponding to the size of pUC18. Lanes 4-8, in thepresence of 25, 50, 75, 100 and 200 μM Ole or HT.C. The effect of Ole and HT on HIV-1 Integrase Disintegration ActivityLeft: Schematic representation of the disintegration activity of HIV-1integrase. The 5′ ³²P-labeled 38-mer dumbbell was used as the substrateand shown with the predicted secondary structure. Disintegration yieldsa ³²P-labeled 14-mer consisting of the viral sequences in the hairpinstem and a 24-mer unlabeled target sequence that has been repaired.Right: Inhibition of disintegration activity of HIV-1 integrase by Oleand HT. Lane 1, the 5′ ³²P-labeled 38-mer dumbbell substrate. Lane 2,treatment with HIV-1 integrase results in the formation of the 5′³²P-labeled 14-mer disintegration product. Lanes 3, 4, 5, 6, 7disintegration assays in the presence of 25, 50, 75, 100, and 200 nMOle. Lanes 8, 9, 10, 11, 12, in the presence of 25, 50, 75, 100, and 200nM HT.

DETAILED DESCRIPTION OF THE INVENTION

Before the present methods and treatment methodology are described, itis to be understood that this invention is not limited to particularmethods, and experimental conditions described, as such methods andconditions may vary. It is also to be understood that the terminologyused herein is for purposes of describing particular embodiments only,and is not intended to be limiting, since the scope of the presentinvention will be limited only in the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein and/or which will become apparent to those persons skilled in theart upon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference.

DEFINITIONS

The terms used herein have the meanings recognized and known to those ofskill in the art, however, for convenience and completeness, particularterms and their meanings are set forth below.

The term “adipocyte” refers to a cell existing in or derived from fattissue which is terminally differentiated. In their differentiatedstate, adipocytes assume a rounded morphology associated withcytoskeletal changes and loss of mobility. They further accumulate lipidas multiple small vesicles that later coalesce into a single, largelipid droplet displacing the nucleus. The term “human adipocyte” refersto an adipocyte existing in or isolated from human fat tissue.Adipocytes play a critical role in energy homeostasis. They synthesizeand store lipids when nutrients are plentiful, and release fatty acidsinto the circulation when nutrients are required. Numerous adipogenicgenes are expressed in functional adipocytes, whereas they are notexpressed in preadipocytes in which lipid are not accumulated either.Adipocyte development has been extensively studied in cell culture aswell as in animal models. There are several lines of evidence supportingthat adipose tissue dysfunction plays an important role in thepathogenesis of type II diabetes mellitus, i.e. failure of adipocytedifferentiation is a predisposition to developing diabetes, (see, e.g.,Danforth (2000) Nature Genetics 26: 13).

The term “adipogenic gene expression” refers to the expression ofseveral genes known as “adipogenesis marker genes” and more particularlyrefers to one or more genes specifically associated with a specificadipogenesis/differentiation stage. Such marker genes are well-known inthe art. For example, Peroxisome Proliferator-Activated Receptor γ2(PPARγ2), lipoprotein lipase (LPL), and the adipocyte-selective fattyacid binding protein (the αP2 gene). In addition, other differentiatedadipocyte marker genes include glycerophosphate dehydrogenase (GPDH),fatty acid synthase, acetyl CoA carboxylase, malic enzyme, Glut 4, andthe insulin receptor (see Spiegelman et al. J. Biol. Chem. 268:6823-6826, 1993, incorporated herein by reference). Preadipocytes alsohave characteristic marker genes, such as the cell surface antigenrecognized by the monoclonal antibody AD-3. Expression level changes ofthe various isoforms of the C/EBP (CCAAT/enhancer-binding proteins)family of transcription factors may also indicate different stages ofadipogenesis (see Yu and Hausman, Exp Cell Res Dec. 15, 1998; 245(2):343-9). Other genes include the lipolytic genes involved in themobilization and 1-oxidation of stored fat as well as those in themitochrondia involved in thermogenesis (the uncoupling enzymes, factorsand modulators) such as PPAR α, PPARδ, and their related genes,hormone-sensitive lipase (HSL), lipoprotein lipase (LPL), and enzymes ofenergy metabolism such as glycerol-3-phosphate dehydrogenase 1,acetyl-coenzyme A carboxykinase, phosphoenolpyruvate carboxykinase andpyruvate dehydrogenase kinase.

The phrase “essentially pure” refers to a cell population, e.g., a humanadipocyte population, that has been isolated from its natural source(e.g., has been isolated or purified from fat tissue, for example, fromhuman fat tissue) and, through a purification step or series ofpurification steps, has been separated from other cells (e.g.,non-adipocyte cells) and cellular debris. An essentially pure cellpopulation, as defined according to the instant invention, is at least90% pure, i.e., at least 90% of the cells are of the desired cell type(e.g., human adipocytes) and less then 10% are contaminating (e.g.non-adipocyte) cells. In a preferred embodiment, an essentially purecell population (e.g., an essentially pure adipocyte population) is atleast 95% pure. In a more preferred embodiment, an essentially pure cellpopulation (e.g., an essentially pure adipocyte population) is at least96%, 97%, 98%, 99% or 100% pure).

The phrase “differentiation-inducing agent” refers to a compound oragent that initiates or stimulates the differentiation of preadipocytesinto adipocytes. Preferred “differentiation-inducing agents” include butare not limited to insulin, insulin-sensitizing agents, substrates forlipid synthesis, PPAR ligands (e.g., natural ligands, for example,prostaglandin J₂, and synthetic ligands, for example,thiazolidinediones, and the like). The phrase “differentiation-promotingagent” refers to a compound or an agent that enhances or accelerates thedifferentiation of preadipocytes into adipocytes. Preferred“differentiation-inducing agents” include but are not limited toinsulin, insulin-sensitizing agents, substrates for lipid synthesis,PPAR ligands, and the like. “Differentiation-inducing agents” or“differentiation-promoting agents” vary considerably in effectivenessbut share common effects on several cellular signaling pathwaysincluding, but not limited to: (1) tyrosine kinase pathways (e.g.,IGF-1-mediated tyrosine kinase pathway); (2) adenylylcyclase/phosphodiesterase signaling pathways; (3)steroid/thyroid/peroxisome proliferator activated (PPAR)/retinoidnuclear receptors signaling pathways; and (4) protein kinase signalingpathways (MacDougald, O. A. et al. (1995) Annu. Rev. Biochem 64:345-73;Smas, C. M. et al. (1995) Biochem J. 309, 697-710; Cornelius, P. et al.(1994) Annu. Rev. Nutr. 14:99-129). Following induction ofdifferentiation through these signal transduction pathways, coordinatedchanges in the expression of over 600 genes occurs leading to theacquisition and maintenance of the fat cell phenotype (MacDougald, O. A.et al. (1995) Annu. Rev. Biochem 64:345-73; Smas, C. M. et al. (1995)Biochem J. 309, 697-710; Cornelius, P. et al. (1994) Annu. Rev. Nutr.14:99-129). These changes in differentiation-dependent gene expressionare orchestrated by several transcription factors including CCAATenhancer binding proteins (C/EBPα, β, and γ), PPARγ, and others(reviewed in MacDougald, O. A. et al. (1995) Annu. Rev. Biochem64:345-73; Smas, C. M. et al. (1995) Biochem J. 309, 697-710; Kirkland,J. L., et al. (1997) J. Amer. Geriatr. Soc. 45:959-67). Overexpressionof some of these transcription factors, including C/EBPα and PPAR γ, issufficient to induce the differentiation of preadipocytes (Lin, F. T.,et al (1994) Proc. Natl. Acad: Sci. USA 91:8757-8761; Hu, E. et al.(1995) Proc. Natl. Acad. Sci. USA 92:8956-60; Wu, Z., et al. (1995)Genes Defer 9:2350-63; Yeh, W. C., et al. (1995) Genes Devel. 9:168-81).

Markers of differentiation include (presented in order of detectablechanges in expression): (1) cytoskeletal genes; (2) lipoprotein lipase(LPL) and collagen isoforms; (3) adipocyte fatty acyl binding protein(aP2) and glycerol-3-phosphate dehydrogenase (G3PD); and/or (4) theinsulin sensitive glucose transporter (GLUT4), angiotensinogen (ang),apolipoprotein E (apoE), leptin, adipsin (complement factor D), proteinC3, factor B, and other genes occur that contribute to theendocrine/paracrine function of adipose tissue. Increased fat cell mass,which is dependent on the balance between rates of adipogenesis,lipogenesis and lipolysis.

“Modulation” or “modulates” or “modulating” refers to up regulation(i.e., activation or stimulation), down regulation (i.e., inhibition orsuppression) of a response, or the two in combination or apart. As usedherein, a fat cell, preadipocyte or adipocyte “modulator” or“modulating” compound or agent is a compound or agent that modulates atleast one biological marker or biological activity characteristic of fatcells and/or fat tissue. The term “modulating” as related to adipocytedifferentiation or adipogenic gene expression, refers to the ability ofa compound or agent to exert an effect on adipocyte differentiation,de-differentiation or transdifferentiation or to alter the expression ofat least one gene (as noted above) related to adipogenesis. In addition,compounds or agents of the invention modulate at least one of (1)differentiation-specific gene expression, (2) lipid metabolism (e.g.,lipogenesis and/or lipolysis), (3) fatty acid uptake, (4) fataccumulation and/or (5) accumulation of cytoplasmic lipid and (6)modulation of PPAR activity.

“Differentiate” or “differentiation” as used herein, generally refers tothe process by which precursor or progenitor cells differentiate intospecific cell types. In the matter of the present invention, the termrefers to the process by which pre-adipocytes become adipocytes.Differentiated cells can be identified by their patterns of geneexpression and cell surface protein expression. The genes associatedwith adipocyte differentiation are noted above. As an example, cells ofadipocyte lineage typically express the following genes: ob, Ucp, PPARγand C/EBPs (see, e.g., Kozak and Kozak, Endocrinology 134(2):906-13(1994)) and Lee et al., J. Clin. Invest. 111 (4): 453-461 (2003). Asused herein, the term “differentiate” refers to having a differentcharacter or function from the original type of tissues or cells. Thus,“differentiation” is the process or act of differentiating.

“Dedifferentiate” or “dedifferentiation” as used herein, refers to theprocess by which lineage committed cells reverse their lineagecommitment and become precursor or progenitor cells. Dedifferentiatedcells can be identified by loss of patterns of gene expression and cellsurface protein expression associated with the lineage committed cells.A loss of expression or decrease in expression levels of one or more ofthe adipocyte genes noted above indicates that an adipocyte hasundergone dedifferentiation.

“Transdifferentiation” refers to the process by which precursor orprogenitor cells pre-committed to cell types of one lineagedifferentiate into specific cell types of another lineage, e.g.,pre-adipocytes can transdifferentiate into osteoblasts and vice versa.Transdifferentiated cells can be identified by their patterns of geneexpression and cell surface protein expression. Typically, cells of anosteoblast lineage express genes such as, for example, alkalinephosphatase, collagen type I, osteocalcin, and osteoponin; and bonespecific transcription factors such as, for example, Cbfa1/Runx2, Osx,gsc, Dlx1, Dlx5, Msx1, Cart1, Hoxa1, Hoxa2, Hoxa3, Hoxb1, rae28, Twist,AP-2, Mf1, Pax1, Pax3, Pax9, TBX3, TBX4, TBX5, and Brachyury (see, e.g.,Olsen et al, 2000 supra and Nakashima et al., Cell 108(1):17-29 (2002).The cells can be transdifferentiated within the same progenitors. Forexample, mesenchymal stem cells or marrow stromal cells (MSC), are stemcells that can differentiate into osteoblasts, chondrocytes, myocytes,adipocytes, neuronal cells, and, as described lately, intobeta-pancreatic islets cells. Thus these cells can be crosstransdifferentiated under optimal culture conditions and/or growthfactors. MSCs cultured in the presence of transformation growth factor(TGF), specifically bone morphogenetic protein (BMP), will differentiateinto chondrocytes, whereas MSCs cultured in serum with ascorbic acid,inorganic phosphate and dexamethasone will differentiate intoosteoblasts. On the other hand, MSCs cultured under adipogenicconditions in the presence of dexamethasone, insulin andisobutyl-methylxanthine will differentiate into adipocytes.

Adipocyte differentiation is a multistep process controlled by theaction of a complex interactive network of transcription factors inmammals. In the fruit fly the serpent gene is critical for the genesisof the fruit fly fat body, which corresponds to mammalian liver andadipose tissue. The GATA family of transcription factors in mice are themammalian homologues of the Drosophila serpent gene. Both GATA-2 andGATA-3 directly bind to specific sites in the proximal promoter of theadipogenic transcription factor, peroxisome proliferaror activatedreceptor-gamma (PPAR-gamma), and negatively regulate its activity. BothGATA-2 and GATA-3 expression are severely defective in the white adiposetissue of several different models of obesity. These results indicatethat GATA-2 and GATA-3 are preadipocyte markers and play an importantrole in adipogenesis.

As used herein, the term “candidate compound” or “candidate therapeutic”or “test compound” or “agent” or “test agent” refers to any compound ormolecule that is to be tested. As used herein, the terms, which are usedinterchangeably, refer to biological or chemical compounds such assimple or complex organic or inorganic molecules, peptides, proteins,antibodies, oligonucleotides, polynucleotides, carbohydrates, orlipoproteins. A vast array of compounds can be synthesized, for exampleoligomers, such as oligopeptides and oligonucleotides, and syntheticorganic compounds based on various core structures, and these are alsoincluded in the terms noted above. In addition, various natural sourcescan provide compounds for screening, such as plant or animal extracts,and the like. Compounds can be tested singly or in combination with oneanother. Agents or candidate compounds can be randomly selected orrationally selected or designed. As used herein, an agent or candidatecompound is said to be “randomly selected” when the agent is chosenrandomly without considering the specific interaction between the agentand the target compound or site. As used herein, an agent is said to be“rationally selected or designed”, when the agent is chosen on anonrandom basis which takes into account the specific interactionbetween the agent and the target site and/or the conformation inconnection with the agent's action. Moreover, the agent may be selectedby its effect on the gene expression profile obtained from screening invitro or in vivo. For example, the gene expression data for primaryhuman preadipocytes and adipocytes can be accessed online throughdatabases including Pub Med, Human Genome Project (HGP), Gene Bank andPDB (Protein Data Bank).

Biochemically, “adipogenesis” is referred to as the process of fat cellformation. It involves commitment, differentiation and maturation ofadipocytes. Adipogenic genes, gene products, enzymes, factors, andpathways are the genes, gene products, enzymes, factors, and pathwaysfor adipocyte differentiation (adipogenesis or fat cell formation), andthese genes are described throughout the present application.“Lipogenesis” is referred to as the process of lipid biosynthesis (fatformation), the conversion of carbohydrate or protein to fat and thesynthesis of triglycerides from 2-monogylcerol and free fatty acids aswell as from glycerol-3-phosphate and fatty acyl CoA. In addition, thesynthesis of long chain fatty acids, using acetyl CoA as the primer andmalonyl CoA as the addition unit also refers to as lipogenesis.Lipogenic genes, gene products, enzymes, factors, and pathways are thegenes, gene products, enzymes, factors, and pathways for lipidbiosynthesis (fat formation), and these genes are also describedthroughout the present application. Thus, adipogenesis is different fromlipogenesis and the two terms are not synonymous. In addition,“adipogenesis” refers to the process whereby adipose tissue, amesodermal derivative, develops from preadipocytes. “Adipogenesis”refers to the process by which an undifferentiated precursor celldifferentiates into an adipocyte (a fat cell, which is a cellcharacterized by the cellular function of fat storage e.g., incytoplasmic lipid droplets). Precursor cells that are involved in theprocess of adipogensis include pre-adipocytes, mesenchymal stem cellsand progenitor cells. Generally, diseases associated with adipogenesisinclude body weight disorders such as obesity and cachexia, andnonshivering and shivering thermogenesis. Accordingly, in one aspect ofthe invention, the agents identified as modulators of adipogenesis arepotentially useful for modulating body weight-related processes,including, for example, treatment of body weight disorders such asobesity and cachexia, and thermogenesis. Treatment of other obesityrelated disorders or conditions are also contemplated. Obesity relateddisorders or conditions may be selected from the group consisting ofcoronary artery disease/cardiovascular disease, hypertension,cerebrovascular disease, stroke, peripheral vascular disease, insulinresistance, glucose intolerance, diabetes mellitus, hyperglycemia,hyperlipidemia, dyslipidemia, hypercholesteremia, hypertriglyceridemia,hyperinsulinemia, atherosclerosis, cellular proliferation andendothelial dysfunction, diabetic dyslipidemia, HIV-relatedlipodystrophy, peripheral vessel disease, cholesterol gallstones,cancer, menstrual abnormalities, infertility, polycystic ovaries,osteoarthritis, sleep apnea, metabolic syndrome (Syndrome X), type IIdiabetes, diabetic complications including diabetic neuropathy,nephropathy, retinopathy, cataracts, heart failure, inflammation,thrombosis, congestive heart failure, and any other cardiovasculardisease related to obesity or an overweight condition. Obesity inducedor related asthma, airway dysfunction and pulmonary disorders are alsoimportant diseases linked to obesity. “Lipolysis” or “lipolytic” refersto the process of lipid degradation/oxidation (fat burning, utilization,energy production). Lipolytic genes, gene products, enzymes, factors,and pathways are the genes, gene products, enzymes, factors, andpathways for lipid oxidation (fat burning, β-oxidation). “Thermogenesis”refers to the process of heat and energy generation, also known asenergy uncoupling. Thermogenic genes, gene products, enzymes, factors,and pathways are the genes, gene products, enzymes, factors, andpathways for body heat generation and energy uncoupling. This processalso increases metabolic rate.

The PPARs play a central role in adipocyte differentiation. PPAR gammaresponsive genes in human adipocyte differentiation are brieflydiscussed below: Affymetrix profiling of gene expression in humanadipocytes identified about 1000 genes that were significantlyup-regulated subsequent to induction of differentiation and 278statistically significantly down-regulated genes [Gene. 2006 Mar. 15;369:90-9]. In addition, it is also necessary to consider PPAR alpha anddelta responsive genes. A recent report (Diabetologia. 2005 September;48(9):1776-83. Epub 2005 Jul. 30) on microarray gene profiling ofisolated abdominal subcutaneous adipocytes from 20 non-obese (BMI 25±3kg/m²) and 19 obese (BMI 55±8 kg/m²) non-diabetic Pima Indians usingAffymetrix HG-U95 GeneChip arrays showed that the most differentiallyexpressed genes in adipocytes of obese individuals consisted of 433upregulated and 244 downregulated genes. Of these, 410 genes could beclassified into 20 functional Gene Ontology categories. The analysesindicated that the inflammation/immune response category wasover-represented, and that most inflammation-related genes wereupregulated in adipocytes of obese subjects. This study providesevidence supporting the active role of mature adipocytes inobesity-related inflammation. It also provides potential candidate genesfor susceptibility to obesity.

Depending on the desired result, an agent identified to induceadipogenesis is potentially useful for increasing body weight and anagent identified to prevent adipogenesis is potentially useful fordecreasing body weight. Adipogenesis in vivo and in vitro is subject tohormonal and transcriptional control, in part mediated by a cascade oftranscription factors including members of the CCAAT/enhancer bindingprotein family, basic helix-loop-helix leucine zipper (bBLH-LZ) family,e.g., ADD1/SREBP1 and peroxisome proliferator activated receptor gamma(PPARgamma) (See, e.g., Wu et al. (1999) Transcriptional activation ofadipogenesis Current Opin. Cell Biol 11:689-694, Rosen and Spiegelman(2000) Molecular regulation of adipogenesis Annu Rev Cell Dev Biol16:145-171, for recent reviews, as well as Kim and Spiegelman (1996)ADD1/SREBP1 promotes adipocyte differentiation and gene expressionlinkedfatty acid metabolism Genes Devel 10:1096-1107). However detailsregarding cellular targets of such transcription factors remain largelyundetermined, as do the mechanisms underlying their action inphysiological and pathological processes.

“Subject” or “patient” refers to a mammal, preferably a human, in needof treatment for a condition, disorder or disease.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human. Preferably, asused herein, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the compound isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water or aqueous solution saline solutions and aqueousdextrose and glycerol solutions are preferably employed as carriers,particularly for injectable solutions. Suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.

A “therapeutically effective amount” or “an effective amount”, which areused interchangeably, is an amount sufficient to decrease or prevent thesymptoms associated with the conditions disclosed herein, includingdiseases associated with obesity, or an amount to inhibit theinfectivity of human immunodeficiency virus (HIV-1 and HIV-2) and otherrelated conditions contemplated for therapy with the compositions of thepresent invention. The effect on HIV infectivity may be measured by anyof the commonly used methods known to those skilled in the art, forexample, cell fusion may be measured by assessing the level of syncitiaformation. Viral replication may be measured by using PCR procedures tomonitor the level of viral nucleic acid in a host cell.

“Metabolic Syndrome” or otherwise known as “Syndrome X” means a diseasecharacterized by spontaneous hypertension, dyslipidemia, insulinresistance, hyperinsulinemia, increased abdominal fat and increased riskof coronary heart disease. As used herein, the terms “Syndrome X”,“Metabolic Syndrome” and “Metabolic Syndrome X” shall mean a disorderthat presents risk factors for the development of Type II diabetesmellitus and cardiovascular disease and is characterized by insulinresistance and hyperinsulinemia and may be accompanied by one or more ofthe following: (a) glucose intolerance, (b) Type II diabetes, (c)dyslipidemia, (d) hypertension and (e) obesity.

“Obesity” refers to a condition in which the body weight of a mammalexceeds medically recommended limits by at least about 20%, based uponage and skeletal size. “Obesity” is characterized by fat cellhypertrophy and hyperplasia. “Obesity” may be characterized by thepresence of one or more obesity-related phenotypes, including, forexample, increased body mass (as measured, for example, by body massindex, or “BMI”), altered anthropometry, basal metabolic rates, or totalenergy expenditure, chronic disruption of the energy balance, increasedFat Mass as determined, for example, by DEXA (Dexa Fat Mass percent),altered maximum oxygen use (VO2), high fat oxidation, high relativeresting rate, glucose resistance, hyperlipidemia, insulin resistance,and hyperglycemia. See also, for example, Hopkinson et al. (1997) An JClin Nutr 65(2): 432-8 and Butte et al. (1999) Am J Clin Nutr 69(2):299-307. “Overweight” individuals are generally having a body mass index(BMI) between 25 and 30. “Obese” individuals or individuals sufferingfrom “obesity” are generally individuals having a BMI of 30 or greater.Obesity may or may not be associated with insulin resistance.

An “obesity-related disease” or “obesity related disorder” or “obesityrelated condition”, which are all used interchangeably, refers to adisease, disorder, or condition, which is associated with, related to,and/or directly or indirectly caused by obesity. The “obesity-relateddiseases”, or the “obesity-related disorders” or the “obesity relatedconditions” include but are not limited to, coronary arterydisease/cardiovascular disease, hypertension, cerebrovascular disease,stroke, peripheral vascular disease, insulin resistance, glucoseintolerance, diabetes mellitus, hyperglycemia, hyperlipidemia,dyslipidemia, hypercholesteremia, hypertriglyceridemia,hyperinsulinemia, atherosclerosis, cellular proliferation andendothelial dysfunction, diabetic dyslipidemia, HIV-relatedlipodystrophy, peripheral vessel disease, cholesterol gallstones,cancer, menstrual abnormalities, infertility, polycystic ovaries,osteoarthritis, sleep apnea, metabolic syndrome (Syndrome X), type IIdiabetes, diabetic complications including diabetic neuropathy,nephropathy, retinopathy, cataracts, heart failure, inflammation,thrombosis, congestive heart failure, and any other cardiovasculardisease related to obesity or an overweight condition and/or obesityrelated asthma, airway and pulmonary disorders.

An individual “at risk” may or may not have detectable disease, and mayor may not have displayed detectable disease prior to the treatmentmethods described herein. “At risk” denotes that an individual who isdetermined to be more likely to develop a symptom based on conventionalrisk assessment methods or has one or more risk factors that correlatewith development of obesity or an obesity-related disease or a diseasefor which PPAR modulation provides a therapeutic benefit, or anindividual who is more likely to acquire HIV through use of sharedneedles or those individuals who may practice unprotected sexualactivity. An individual having one or more of these risk factors has ahigher probability of developing obesity or an obesity-related disease,or HIV than an individual without these risk factors. Examples (i.e.,categories) of risk groups are well known in the art and discussedherein.

“Development” or “progression” of obesity herein means initialmanifestations and/or ensuing progression of the disorder. Developmentof obesity can be detectable and assessed using standard clinicaltechniques, such as measurement of increased body mass (as measured, forexample, by body mass index, or “BMI”), altered anthropometry, basalmetabolic rates, or total energy expenditure, chronic disruption of theenergy balance, increased Fat Mass as determined, for example, by DEXA(Dexa Fat Mass percent), altered maximum oxygen use (VO2), high fatoxidation, high relative resting rate, glucose resistance,hyperlipidemia, insulin resistance, and hyperglycemia. See also, forexample, Hopkinson et al. (1997) Am J Clin Nutr 65(2): 432-8 and Butteet al. (1999) Am J Clin Nutr 69(2): 299-307. However, development alsorefers to disease progression that may be undetectable. For purposes ofthis invention, development or progression refers to the biologicalcourse of the disease state. “Development” includes occurrence,recurrence, and onset. As used herein “onset” or “occurrence” of obesityincludes initial onset and/or recurrence.

As used herein, “delaying development” of obesity, or an obesity-relateddisease, means to defer, hinder, slow, retard, stabilize, and/orpostpone development of the disease. This delay can be of varyinglengths of time, depending on the history of the disorder and/or themedical profile of the individual being treated. As is evident to oneskilled in the art, a sufficient or significant delay can, in effect,encompass prevention, in that the individual does not develop detectabledisease. A method that “delays” development of disease is a method thatreduces the extent of the disease in a given time frame, when comparedto not using the method. Such comparisons are typically based on animalor clinical studies, using a statistically significant number ofsubjects, although this knowledge can be based upon anecdotal evidence.“Delaying development” can mean that the extent and/or undesirableclinical manifestations are lessened and/or time course of theprogression is slowed or lengthened, as compared to not administeringthe agent. Thus the term also includes, but is not limited to,alleviation of symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, delay or slowing of diseaseprogression, and remission (whether partial or total) whether detectableor undetectable.

“Diabetes” refers to high blood sugar or ketoacidosis, as well aschronic, general metabolic abnormalities arising from a prolonged highblood sugar status or a decrease in glucose tolerance. “Diabetes”encompasses both the type I and type II (Non Insulin Dependent DiabetesMellitus or NIDDM) forms of the disease. The risk factors for diabetesinclude the following factors: waistline of more than 40 inches for menor 35 inches for women, blood pressure of 130/85 mmHg or higher,triglycerides above 150 mg/dl, fasting blood glucose greater than 100mg/dl or high-density lipoprotein of less than 40 mg/dl in men or 50mg/dl in women.

The term “hyperinsulinemia” refers to a state in an individual in whichthe level of insulin in the blood is higher than normal.

The term “insulin resistance” refers to a state in which a normal amountof insulin produces a subnormal biologic response relative to thebiological response in a subject that does not have insulin resistance.

An “insulin resistance disorder” as discussed herein, refers to anydisease or condition that is caused by or contributed to by insulinresistance. Examples include: diabetes, obesity, metabolic syndrome,insulin-resistance syndromes, syndrome X, insulin resistance, high bloodpressure, hypertension, high blood cholesterol, dyslipidemia,hyperlipidemia, dyslipidemia, atherosclerotic disease including stroke,coronary artery disease or myocardial infarction, hyperglycemia,hyperinsulinemia and/or hyperproinsulinemia, impaired glucose tolerance,delayed insulin release, diabetic complications, including coronaryheart disease, angina pectoris, congestive heart failure, stroke,cognitive functions in dementia, retinopathy, peripheral neuropathy,nephropathy, glomerulonephritis, glomerulosclerosis, nephrotic syndrome,hypertensive nephrosclerosis some types of cancer (such as endometrial,breast, prostate, and colon), complications of pregnancy, poor femalereproductive health (such as menstrual irregularities, infertility,irregular ovulation, polycystic ovarian syndrome (PCOS)), lipodystrophy,cholesterol related disorders, such as gallstones, cholescystitis andcholelithiasis, gout, obstructive sleep apnea and respiratory problems,osteoarthritis, and prevention and treatment of bone loss, e.g.osteoporosis.

“Alpha-glucosidase inhibitors” act to inhibit alpha-glucosidase, whichis an enzyme that converts fructose to glucose, thus these inhibitorsdelay the digestion of carbohydrates. The undigested carbohydrates aresubsequently broken down in the gut, thereby reducing the post-prandialglucose peak. Suitable examples include, but are not limited to,acarbose, voglibose and miglitol.

“Sulfonylureas” increase insulin production by stimulating pancreaticbeta cells, and therefore act as insulin secretagogues. The primarymechanism of action of sulfonylureas is to close ATP-sensitive potassiumchannels in the beta-cell plasma membrane, initiating a chain of eventsthat result in insulin release. Suitable examples of sulfonylureasinclude, but are not limited to chlorpropamide, tolazamide, tolbutamide,glyburide, glipizide, glimepiride, and like. Meglitinides, another classof insulin secretagogues, that have a mechanism of action distinct fromthat of the sulfonylureas. Suitable examples of meglitinides include,but are not limited to repaglinide. Agents which modify insulinsecretion such as Glucagon-like Peptide-1 (GLP-1) and it's mimetics,Glucose-insulinotropic peptide (GIP) and it's mimetics, Exendin and it'smimetics, and Dipeptyl Protease Inhibitors (DPPIV) are also contemplatedfor use with the invention.

“Biguanides” are compounds that decrease liver glucose production andincrease the uptake of glucose. Suitable examples include, but are notlimited to metformin, phenformin and buformin.

A “peroxisome proliferator activated receptor” or “PPAR” is a member ofa family of nuclear receptors, distinguished in alpha (α), delta (6),and gamma (γ) subtypes as described herein. As used herein the term“PPAR” refers to a peroxisome proliferator-activated receptor asrecognized in the art. As indicated above, the PPAR family includes PPARα (also referred to as PPARa or PPARalpha), PPAR δ (also referred to asPPAR beta, PPARd or PPARdelta), and PPAR γ (also referred to as PPARg orPPARgamma). The individual PPARs can be identified by their sequences,where exemplary reference sequence accession numbers are: NM_(—)005036(cDNA sequence for hPPARa (SEQ ID NO:1)), NP_(—)005027 (protein sequencefor hPPARa (SEQ ID NO: 2)), NM_(—)015869 (cDNA sequence for hPPARgisoform 2 (SEQ ID NO: 3)), NP_(—)056953 (protein sequence for hPPARgisoform 2 (SEQ ID NO: 4)), NM_(—)006238 (cDNA sequence for hPPARd (SEQID NO: 5)), and NP_(—)006229 (protein sequence for hPPARd (SEQ ID NO:6)). One of ordinary skill in the art will recognize that sequencedifferences will exist due to allelic variation, and will also recognizethat other animals, particularly other mammals have corresponding PPARs,which have been identified or can be readily identified using sequencealignment and confirmation of activity, can also be used. One ofordinary skill in the art will also recognize that modifications can beintroduced in a PPAR sequence without destroying PPAR activity. Suchmodified PPARs can also be used in the present invention, e.g., if themodifications do not alter the binding site conformation to the extentthat the modified PPAR lacks substantially normal ligand binding.

A “PPAR modulating agent” or “PPAR modulator” refers to any agent thatbinds to any of the PPAR receptors and acts to either enhance theactivity or function of that particular receptor (agonist) or acts toinhibit or depress the activity or function of that particular receptor(antagonist). The PPAR receptors include the α, γ, and δ receptors. Itis also possible to have the following types of PPAR modulators:

-   -   Selective PPAR modulators that serve as selective or partial        agonist/antagonist and uncouple therapeutic effects from adverse        side effects. For example, a selective/partial PPAR γ        agonist/antagonist that uncouples the therapeutic effect of        insulin sensitizing activity from adipogenic effect (side effect        for weight gain during treatment)    -   Dual PPAR modulators that act on two PPARs collectively,        selectively or partially either as agonists or        agonist-antagonists combined. For example, the dual PPARγ/PPARα        agonists have two separate therapeutic targets in metabolic        pathways in adipose tissue and liver. They may improve both        hyperglycemia and atherogenic dyslipidaemia and may further        reduce the inflammatory component of atherogenesis.    -   Pan PPAR modulators that interact with all of the three PPARs        collectively, selectively or partially either as agonists or        agonist-antagonists combined. For example, the pan        PPARγ/PPARα/PPAR δ agonists have three separate therapeutic        targets—metabolic pathways in adipose tissue, liver and muscle        (and other tissue). They may improve hyperglycemia, atherogenic        dyslipidemia, inflammatory component of atherogenesis, energy        uncoupling and weight reduction.

“Thiazolidinediones” are insulin sensitizing drugs, which decreaseperipheral insulin resistance by enhancing the effects of insulin attarget organs and tissues. These drugs bind and activate the nuclearreceptor, peroxisome proliferator-activated receptor-gamma (PPAR-gamma)which increases transcription of specific insulin-responsive genes.Suitable examples of PPAR-gamma agonists are the thiazolidinedioneswhich include, but are not limited to rosiglitazone, pioglitazone,troglitazone, isaglitazone (known as MCC-555),2-[2-[(2R)-4-hexyl-3,4-dihydro-3-oxo-2H-1,4-benzoxazin-2-yl]ethoxy]-benzeneacetic acid, and the like. Additionally, the non-thiazolidinediones alsoact as insulin sensitizing drugs, and include, but are not limited toGW2570, and the like.

The concept of “combination therapy” is well exploited in currentmedical practice. Treatment of a pathology by combining two or moreagents that target the same pathogen or biochemical pathway sometimesresults in greater efficacy and diminished side effects relative to theuse of the therapeutically relevant dose of each agent alone. In somecases, the efficacy of the drug combination is additive (the efficacy ofthe combination is approximately equal to the sum of the effects of eachdrug alone), but in other cases the effect can be synergistic (theefficacy of the combination is greater than the sum of the effects ofeach drug given alone). As used herein, the term “combination therapy”means the two compounds can be delivered in a simultaneous manner, e.g.concurrently, or wherein one of the compounds is administered first,followed by the second agent, e.g sequentially. The desired result canbe either a subjective relief of one or more symptoms or an objectivelyidentifiable improvement in the recipient of the dosage.

“Atherogenesis” is a process of forming atheromas, that is, plaques inthe inner lining (the intima) of arteries. It is a process in which theimmune system appears to take an active part, and refers to the build-upof plaque in the blood vessels. Accordingly, activated lymphocytes havebeen detected in human and murine plaques, sometimes even preceding theinfiltrating lipid-laden macrophages. The atherosclerotic processentails a proliferative phenotype, involving, apart from lymphocytes andmacrophages, also smooth muscle cells, which occupy lesions that arerelatively more advanced. Furthermore, atherosclerosis is theaccumulation of lipid, inflammatory cells, and fibrous tissue in theintima, which causes intimal thickening of large and mid-sized arteries.The clinical manifestations differ depending on the circulatory bedaffected. The coronary arteries are particularly susceptible toatherogenesis; atherosclerosis of the coronary arteries may lead toangina pectoris and myocardial infarction. Dyslipidemia is a primary,major risk factor for coronary artery disease (CAD) and may even be aprerequisite for CAD, occurring before other major risk factors comeinto play. “Diet-induced atherogenesis” refers to the induction orinitiation of the atherogenic process by consumption of food thatcontains a high fat content, which ultimately results in build-up ofplaque in the blood vessels.

“Endothelial dysfunction” is a physiological dysfunction of normalbiochemical processes carried out by endothelial cells, the cells thatline the inner surface of all blood vessels, arteries and veins.Compromise of normal function of endothelial cells is characteristic ofendothelial dysfunction. Normal functions of endothelial cells includemediation of coagulation, platelet adhesion, immune function, control ofvolume and electrolyte content of the intravascular and extravascularspaces. An important consequence of endothelial dysfunction is theinability of a vessel to dilate in response to physiological stimuli,such as increases in blood flow, reflecting impaired flow-dependent,endothelium-mediated vasodilation (FDD). Accumulating evidence suggeststhat endothelial dysfunction contributes to exercise intolerance,impaired myocardial perfusion, and left ventricular remodelling incongestive heart failure. Moreover, impaired endothelial function isassociated with a number of disease states, including cardiovasculardisease (CVD) and its major risk factors. Endothelial dysfunctionprecedes overt vascular disease by years and may itself be a potentiallymodifiable CVD risk factor. Although no gold standard for themeasurement of endothelial function exists, the measurement offlow-mediated dilation (FMD) in the brachial artery, assessed withDoppler ultrasonography, is the most studied method and shows the mostpromise for clinical application. It is a well-tolerated, noninvasive,and low-risk procedure. Brachial artery FMD is an attractive screeningtool for assessing endothelial dysfunction.

“Treatment” or “treating” refers to therapy, prevention and prophylaxisand particularly refers to the administration of medicine or theperformance of medical procedures with respect to a patient, for eitherprophylaxis (prevention) or to cure (if possible) or reduce the extentof or likelihood of occurrence of the infirmity or malady or conditionor event in the instance where the patient is afflicted. Moreparticularly, as related to the present invention, “treatment” or“treating” is defined as the application or administration of atherapeutic agent to a patient, or application or administration of atherapeutic agent to an isolated tissue or cell line from a patient, whohas a disease, a symptom of disease or a predisposition towarddevelopment of a disease. Treatment can slow, cure, heal, alleviate,relieve, alter, remedy, ameliorate, improve or affect the disease, asymptom of the disease or the predisposition toward disease, e.g., by atleast 10%. In the present invention, the treatments using the agentsdescribed may be provided to slow or halt weight gain, or to aid inweight reduction, or to prevent fat accumulation or obesity, or toinhibit adipocyte differentiation or adipogenic gene expression, or toincrease the amount or quality of lean muscle mass present in themammal, or to prevent the infectivity of HIV, or to slow viral spread,or to alleviate one or more symptoms associated with the presence of theviral disease. More preferably, the goal is the treatment of obesity orobesity-related diseases, disorders or conditions and the morbidityassociated with such conditions and the treatment of humanimmunodeficiency virus infections and the symptoms and sequelaeassociated with the presence of the viral infection.

“Diagnosis” or “screening” refers to diagnosis, prognosis, monitoring,characterizing, selecting patients, including participants in clinicaltrials, and identifying patients at risk for or having a particulardisorder or clinical event or those most likely to respond to aparticular therapeutic treatment, or for assessing or monitoring apatient's response to a particular therapeutic treatment.

A “small molecule” refers to a composition that has a molecular weightof less than 3 kilodaltons (kDa), and preferably less than 1.5kilodaltons, and more preferably less than about 1 kilodalton. Smallmolecules may be nucleic acids, peptides, polypeptides, peptidomimetics,carbohydrates, lipids or other organic (carbon-containing) or inorganicmolecules. As those skilled in the art will appreciate, based on thepresent description, extensive libraries of chemical and/or biologicalmixtures, often fungal, bacterial, or algal extracts, may be screenedwith any of the assays of the invention to identify compounds thatmodulate a bioactivity. A “small organic molecule” is an organiccompound (or organic compound complexed with an inorganic compound(e.g., metal)) that has a molecular weight of less than 3 kilodaltons,and preferably less than 1.5 kilodaltons, and more preferably less thanabout 1 kilodalton.

“Disease associated with PPAR γ, δ, or α” or “a disease associated witha PPAR γ, δ, or α receptor” includes diseases treatable with a ligand ofany of the above-noted PPAR receptors, such as but not limited to TypeII diabetes and obesity, or other obesity related disorders. Diseasesrelated to PPAR expression and/or activity would also be considered asassociated with the PPAR receptor, such as obesity or other disordersexpected to be affected by alterations in the PPAR's role in adipocytedifferentiation, de-differentiation and transdifferentiation as well asadipocyte functions including fat synthesis, storage, utilization andenergy uncoupling. The diseases associated with the modulation of thedifferent PPAR receptors are known to those skilled in the art. Forexample, possible diseases and/or risk factors associated with PPARsare: the metabolic syndrome and its associated risk factors foratherosclerotic cardiovascular disease (ASCVD) and diabetes, atherogenicdyslipidemia, elevated blood pressure, elevated plasma glucose, aprothrombotic state and a pro-inflammatory state. In addition, otherconditions, including fatty liver, polycystic ovary disease, sleepapnea, cholesterol gallstones, asthma and cancer.

“Prophylactic” or “therapeutic” treatment refers to administration tothe host of one or more of the subject compositions. If it isadministered prior to clinical manifestation of the unwanted condition(e.g., disease or other unwanted state of the host animal) then thetreatment is prophylactic, i.e., it protects the host against developingthe unwanted condition, whereas if administered after manifestation ofthe unwanted condition, the treatment is therapeutic (i.e., it isintended to diminish, ameliorate or maintain the existing unwantedcondition or side effects therefrom).

“Therapeutic agent” or “therapeutic” refers to an agent capable ofhaving a desired biological effect on a host. Chemotherapeutic andgenotoxic agents are examples of therapeutic agents that are generallyknown to be chemical in origin, as opposed to biological, or cause atherapeutic effect by a particular mechanism of action, respectively.Examples of therapeutic agents of biological origin include growthfactors, hormones, and cytokines. A variety of therapeutic agents areknown in the art and may be identified by their effects. Certaintherapeutic agents are capable of regulating cell proliferation anddifferentiation. Examples include chemotherapeutic nucleotides, drugs,hormones, non-specific (non-antibody) proteins, oligonucleotides (e.g.,antisense oligonucleotides that bind to a target nucleic acid sequence(e.g., mRNA sequence)), peptides, and peptidomimetics. Examples of othertherapeutic agents that modulate PPAR gamma, alpha and delta are thethiazolidinediones, firbrates, fatty acids and eicosanoids.

“Therapeutic effect” refers to a local or systemic effect in animals,particularly mammals, and more particularly humans caused by apharmacologically active substance. The term thus means any substanceintended for use in the diagnosis, cure, mitigation, treatment orprevention of disease or in the enhancement of desirable physical ormental development and conditions in an animal or human.

“Agonist” refers to an agent that mimics or up-regulates (e.g.,potentiates or supplements) the bioactivity of a protein. An agonist maybe a wild-type protein or derivative thereof having at least onebioactivity of the wild-type protein. An agonist may also be a compoundthat up-regulates expression of a gene or which increases at least onebioactivity of a protein. An agonist may also be a compound whichincreases the interaction of a polypeptide with another molecule, e.g.,a target peptide or nucleic acid. An agonist may also be a compound thatincrease or up-regulate the activity and/or function of a protein,peptide, an enzyme or biofactor.

“Antagonist” refers to an agent that down-regulates (e.g., suppresses orinhibits) at least one bioactivity of a protein. An antagonist may be acompound which inhibits or decreases the interaction between a proteinand another molecule, e.g., a target peptide or enzyme substrate. Anantagonist may also be a compound that down-regulates expression of agene or which reduces the amount of expressed protein present. Anantagonist may also be a compound that decrease or down-regulate theactivity and/or function of a protein, peptide, an enzyme or biofactor.

“Analog” or “analogue” as used herein, refers to a chemical compound, anucleotide, a protein, or a polypeptide that possesses similar oridentical activity or function(s) as the chemical compounds,nucleotides, proteins or polypeptides having the desired activity andtherapeutic effect of the present invention (eg. to inhibit fataccumulation, or to modulate adipocyte differentiation or adipogenicgene expression or to treat obesity or obesity-related diseases,disorders or conditions), but need not necessarily comprise a compoundthat is similar or identical to those compounds of the preferredembodiment, or possess a structure that is similar or identical to theagents of the present invention. An agent having activity “analogous tooleuropein” is an agent that possesses similar functions and activitiesas oleuropein, including the effects on the adipogenic and lipolyticgenes described herein, and on adipocyte metabolism, differentiation,de-differentiation, or transdifferentiation or effects on the PPARreceptors described herein.

“Derivative” refers to the chemical modification of molecules, eithersynthetic organic molecules or proteins, nucleic acids, or any class ofsmall molecules such as fatty acids, or other small molecules that areprepared either synthetically or isolated from a natural source, such asa plant, that retain at least one function of the active parentmolecule, but may be structurally different. Chemical modifications mayinclude, for example, replacement of hydrogen by an alkyl, acyl, oramino group. It may also refer to chemically similar compounds whichhave been chemically altered to increase bioavailability, absorption, orto decrease toxicity. A derivative polypeptide is one modified byglycosylation, pegylation, or any similar process that retains at leastone biological or immunological function of the polypeptide from whichit was derived.

As used herein in connection with the design or development of PPARligands, the term “bind” and “binding” and like terms refer to anon-convalent energetically favorable association between the specifiedmolecules (i.e., the bound state has a lower free energy than theseparated state, which can be measured calorimetrically). For binding toa target, the binding is at least selective, that is, the compound bindspreferentially to a particular target or to members of a target familyat a binding site, as compared to non-specific binding to unrelatedproteins not having a similar binding site. For example, BSA is oftenused for evaluating or controlling for non-specific binding. Inaddition, for an association to be regarded as binding, the decrease infree energy going from a separated state to the bound state must besufficient so that the association is detectable in a biochemical assaysuitable for the molecules involved.

By “binding pocket” is meant a specific volume within a binding site. Abinding pocket is a particular space within a binding site at leastpartially bounded by target molecule atoms. Thus a binding pocket is aparticular shape, indentation, or cavity in the binding site. Bindingpockets can contain particular chemical groups or structures that areimportant in the non-covalent binding of another molecule such as, forexample, groups that contribute to ionic, hydrogen bonding, van derWaals, or hydrophobic interactions between the molecules.

In the context of target molecules in the present invention, the term“crystal” refers to an ordered complex of target molecule, such that thecomplex produces an X-ray diffraction pattern when placed in an X-raybeam. Thus, a “crystal” is distinguished from a disordered or partiallyordered complex or aggregate of molecules that do not produce such adiffraction pattern. Preferably a crystal is of sufficient order andsize to be useful for X-ray crystallography. A crystal may be formedonly of target molecule (with solvent and ions) or may be a co-crystalof more than one molecule, for example, as a co-crystal of targetmolecule and binding compound, and/or of a complex of proteins (such asa holoenzyme).

By “designing a ligand”, “preparing a ligand”, “discovering a ligand”,and like phrases is meant the process of considering relevant data(especially, but not limited to, any individual or combination ofbinding data, X-ray co-crystallography data, molecular weight, clogP,and the number of hydrogen bond donors and acceptors) and makingdecisions about advantages that can be achieved with resort to specificstructural modifications to a molecule, and implementing thosedecisions. This process of gathering data and making decisions aboutstructural modifications that can be advantageous, implementing thosedecisions, and determining the result can be repeated as many times asnecessary to obtain a ligand with desired properties.

By “docking” is meant the process of attempting to fit athree-dimensional configuration of a binding pair member into athree-dimensional configuration of the binding site or binding pocket ofthe partner binding pair member, which can be a protein, and determiningthe extent to which a fit is obtained. The extent to which a fit isobtained can depend on the amount of void volume in the resultingbinding pair complex (or target molecule-ligand complex). Theconfiguration can be physical or a representative configuration of thebinding pair member, e.g., an in silico representation or other model.

In the context of development of modulators using molecular scaffolds,by “ligand” is meant a molecular scaffold that has been chemicallymodified at one or more chemically tractable structures to bind to thetarget molecule with altered or changed binding affinity or bindingspecificity relative to the molecular scaffold. The ligand can bind witha greater specificity or affinity for a member of the molecular familyrelative to the molecular scaffold. A ligand binds non-covalently to atarget molecule, which can preferably be a protein or enzyme.

By “orientation”, in reference to a binding compound bound to a targetmolecule is meant the spatial relationship of the binding compound andat least some of its consitituent atoms to the binding pocket and/oratoms of the target molecule at least partially defining the bindingpocket.

Binding compounds can be characterized by their affinity for the targetmolecule as measured by determining the dissociation constant or bymeasuring their effect on the activity of the target molecule. Forexample, the IC50 (or EC50) is defined as the concentration of compoundat which 50% of the activity of the target molecule (e.g., enzyme orother protein) activity being measured is lost (or gained) relative toactivity when no compound is present. Activity can be measured usingmethods known to those of ordinary skill in the art, e.g., by measuringany detectable product or signal produced by occurrence of an enzymaticreaction, or other activity by a protein being measured. For PPARagonists, activities can be determined as described in the Examples, orusing other such assay methods known in the art.

In addition, the interaction of a ligand with its target structure canbe assessed by measurement of the free energy in kcal/mol usingapproaches that involve AutoDock, Molecular Dynamics (MD) and MolecularMechanics/Poisson-Boltzmann Solvent Accessible surface area (MM-PBSA)calculations. This is done using the crystal structures of the ligandbinding domains of the PPAR receptors. Molecular docking is used togenerate several distinct binding orientations. Molecular dynamicssimulation is used to further relax the complex. MM-PBSA is then used toestimate the affinity for each binding mode. The binding modes with thelowest free energy are expected to be the most favorable. Theenergetically most favorable binding mode would provide for a freeenergy of <0 kcal/mol (negative value), and larger the free energy, theless favorable the interaction. The binding between a ligand and itstarget is unique in each system and can not be compared to othersystems. However, it can be stated that the smaller the free energy themore favorable the binding. For favorable interactions, the free energyis negative. The more negative, the more favorable the interaction is.

The MM/PBSA approach uses a set of structures collected with moleculardynamics to evaluate free energies of binding. This method combines themolecular mechanical energies with the continuum solvent approaches. Themolecular mechanical energies are determined based on AMBER force fieldand represent the internal energy (bond, angle and dihedral), van derWaals and electrostatic interactions. The electrostatic contribution tothe solvation free energy is calculated with the Poisson-Boltzmann (PB)method. The hydrophobic contribution to the solvation free energy isdetermined with solvent-accessible-surface-area dependent terms. Andestimates of conformational entropies can be made with the mode modulefrom AMBER. Briefly, MM/PBSA methodology involves the following steps:

1. Perform short molecular dynamics simulation.

2. Calculate average molecular mechanical and solvation energies from aset of simulation snapshots.

3. Use the quasi-harmonic approximation to/approximate/the entropy ofthe system.

By putting energy terms together, free energy of binding can beestimated with the formula

ΔG≅U(EE)+ΔG(solv)−TΔS

Where ΔG=standard free energy (kcal/mol), U (EE)=MM energy (kcal/mol),ΔG (solv)=free energy of solvent (kcal/mol), T=temperature (Kelvin) andΔS=entropy (kcal/mol K).

“Resistin” (resistance to insulin) or “Resistin-Like Molecules” “RELMs”or “FIZZ1-3” are found in the inflammatory zone involved in allegory andinflammation. Resistin is an adipokine associated with obesity and type2 diabetes. Serum resistin level is related to insulin resistance.(Degawa-Yamauchi M, Serum Resistin (FIZZ3) Protein Is Increased in ObeseHumans, J. Clin. Endocrinol. Metab., 88: 5452-5455, (2003); Anal Chem.(2006) May 15; 78(10):3271-6)

“Adipocyte-Specific Secretory Factor” (ADSF) is a small cysteine-richprotein secreted from adipose tissue (adipocyte-derived hormones,adipokines) that belongs to a gene family found in inflammatory zone(FIZZ) or found in resistin-like molecule (RELM). ADSF has beenimplicated in modulating adipogenesis and insulin resistance. Itinhibits adipogenesis, decreases plasma triglyceride and free fattyacid, improves glucose tolerance and insulin sensitivity. (Endocrine.(2006) February; 29(1):81-90; Proc Natl Acad Sci USA. (2004) April 27;101(17):6780-5. Epub (2004) April 16).

“Leptin” is a 16 kDa adipokine (protein hormone), encoded by the obese(ob) gene, expressed predominantly by adipocytes. It is important inregulating body weight, metabolism and reproductive function. Smalleramounts of leptin are also secreted by cells in the epithelium of thestomach and in the placenta. Leptin receptors are highly expressed inareas of the hypothalamus known to be important in regulating bodyweight, as well as in T lymphocytes and vascular endothelial cells (IntJ Obes (Lond). (2006) May 16)

“Acrp30/Adiponectin” (adipocyte complement-related protein of 30 kDa)also known as AdipoQ, APM1, Adiponectin, Gelatin binding protein 28kDa/GBP28 or adipocyte most abundant gene transcript is identified as anovel adipocyte-specific synthesized and secreted protein withstructural resemblance to complement factor C1q. Like adipsin, Acrp30secretion is induced ˜10-fold during adipocyte differentiation. Plasmalevels are reduced in obese humans, and low levels are associated withinsulin-resistance. (Endocrinology. (2006) June; 147(6):2690-5. Epub(2006) March 2). Adiponectin is an adipokine with insulin-sensitizing,anti-inflammatory, and anti-atherogenic properties. Plasma levels ofadiponectin are reduced in insulin resistant states such as obesity,type 2 diabetes and cardiovascular disease. (Hum Reprod. (2006) May 12;[Epub ahead of print]; Biochem Biophys Res Commun. (2006) June 23;345(1):332-9. Epub (2006) April 27. J Endocrinol Invest. (2006) March;29(3):231-6).

“Orexins” is a neuropeptide that stimulates appetite (Br J Nutr. (2004)August; 92 Suppl 1:S47-57)

“Glucose Transporter” (Glut1-Glut14) “Glut 4” is a glucose transporterexpressed uniquely in muscle and fat tissues. It moves fromintracellular sites to the plasma membrane following insulin stimulationand thus increases the rate of glucose transport into these tissues.GLUT4 expression is absent or low in most cultured cell models, which isa disadvantage for studies of glucose metabolism. However, inconditionally immortalized muscle there is a clear rise in GLUT4 levelsfollowing differentiation. (Clin Exp Pharmacol Physiol. 2006 April;33(4):395-9; Am J Med. 2006 May; 119(5 Suppl 1):S10-6).

“Hypoxia-inducible factor-1” (HIF-1) is a transcription factor. It playsa critical role in the transduction of the metabolic response tohypoxia. HIF-1 is composed of α and β subunits, the β-subunit isexpressed constitutively whereas the α-subunit is induced by hypoxia.HIF-1 is activated in hypoxia by the stimulation of the expression ofthe HIF-1α subunit to form the functional transcription factor. HIF-1are expressed in adipocytes and hypoxia cell culture leads to increasedlevels of adipokines. Insulin activates hypoxia-inducible factor-1 alphain human vascular smooth muscle cells via phosphatidylinositol-3 kinaseand mitogen-activated protein kinase pathways (HIF-alpha, beta,Diabetologia. (2006) May; 49(5):1049-63. Epub (2006) February 28;Biochem Biophys Res Commun. (2006) March 10; 341(2):549-56.) Am JPhysiol Endocrinol Metab. (2006) March; 290(3):E591-7. Epub 2005 Oct. 18

“Pref-1” or Preadipocyte factor-1 is a transmembrane protein withepidermal growth factor-like domain. It is highly expressed inpreadipocytes. Pref-1 expression is, however, completely abolished inadipocytes.

“Adipsin” is a serine protease that is secreted by adipocytes. It isdeficient in several animal model of obesity. Adipsin has now beenidentified as the same protein as complement factor D or C3 convertaseactivator or properdin factor D. Its expression is induced upondifferentiation of preadipocytes.

GENERAL DESCRIPTION

Olive leaf has a number of constituents, including oleuropein andseveral types of flavinoids, including rutin, apigenin, luteolin. In thestudies presented herein, “ole” refers to oleuropein, and “OLE” refersto Olive Leaves Extract. Recent studies have centered on oleuropein(ole), which is a non-toxic glucoside isolated from olive leaves, whichhas been shown to have a number of beneficial medicinal properties. Forexample, early studies by Fleming et al. demonstrated the anti-microbialeffects of oleuropein (Fleming, H. P. et al. (1973), Applied Microbiol.26: 777-782). This work was further supported by the studies ofZanichelli, et al. (Zanichelli, D. et al. (2005) J. Food Prot.68(7):1492-1496) and Micol et al. (Micol, V. et al. (2005), AntiviralRes. 66(2-3): 129-136). Carluccio et al. have shown in vitro thatoleuropein inhibits endothelial adhesion molecule expression (Carluccio,M. A. et al. (2003) Arterioscler Thromb Vasc Biol. 23: 622-629). Hamdiet al. have shown that oleuropein has anti-tumor properties and acts todisrupt actin filaments in cells in culture (Hamdi, H. K. et al. (2005)Biochem Biophys. Res. Commun. July 14 Epub). Miles et al. demonstratedthat oleuropein inhibits certain inflammatory cytokines (Miles, E. A. etal. (2005), Nutrition, 21(3): 389-394). Further studies by Manna, et al.demonstrated that oleuropein prevents ischemia and reperfusion inducedmyocardial injury (Manna C. et al. (2004) J. Nutr. Biochem. 15(8):461-466). Studies by Puel et al. demonstrated that oleuropein preventedinflammation-induced osteopenia in ovariectomised rats (Puel, C. et al.(2004) 92(1): 119-127). Earlier studies by Visioli et al. demonstratedthat oleuropein protects low density lipoprotein from CuSO4-inducedoxidation. Coni et al (2000) have subsequently demonstrated thatoleuropein can increase the ability of low density lipoprotein to resistoxidation in vivo (Coni E. et al. (2000), Lipids, 35(1): 45-54), whileCaruso et al. have also shown that oleuropein can prevent cholesteroloxide formation in vitro (Caruso, D. (1999) Nutr. Metab. Cardiovasc.Dis. 9(3):102-107).

Oleuropein is reported herein to be a novel modulator of adipocytedifferentiation, de-differentiation, trans-differentiation, andadipogenic and lipolytic genes and gene products expression and alsodecreases in fat accumulation and increases in fat burning. Theoleuropein was prepared from olive leave extract as reported inLee-Huang et al. (Lee-Huang S; Zhang L; Huang P L; Chang Y T; Huang P L.“Anti-HIV activity of olive leaf extract (OLE) and modulation of hostcell gene expression by HIV-1 infection and OLE treatment”. Biochemical& biophysical research communications. 2003; 307:1029); 307:1029.Commercial oleuropein was used as a standard for the comparison with theoleuropein preparations used in the present studies. In addition,oleuropein has been found to be effective in modulation of endothelialdysfunction and to reduce diet induced atherosclerosis. Moreover,oleuropein and derivatives or analogues thereof reduce the number of fatcells by modulating adipocyte differentiation, de-differentiation andtrans-differentiation. Likewise, oleuropein and its derivatives oranalogues thereof reduce fat accumulation, decrease the size of the fatcells, increase fat burning and expenditure by modulating adipocytemetabolism in terms of down-regulation of lipogenesis (fat formation),up-regulation of lipolysis (fat burning) and thermogenesis viamodulating the lipogenic, lipolytic and thermogenic genes, geneproducts, enzymes, factors, and pathways. Accordingly, based on thefindings presented here, oleuropein is proposed to be useful fortreating obesity or obesity related disorders, such as, but not limitedto, coronary artery disease/cardiovascular disease, hypertension,cerebrovascular disease, stroke, peripheral vascular disease, insulinresistance, glucose intolerance, diabetes mellitus, hyperglycemia,hyperlipidemia, dyslipidemia, hypercholesteremia, hypertriglyceridemia,hyperinsulinemia, atherosclerosis, cellular proliferation andendothelial dysfunction, diabetic dyslipidemia, HIV-relatedlipodystrophy, peripheral vessel disease, cholesterol gallstones,cancer, menstrual abnormalities, infertility, polycystic ovaries,osteoarthritis, sleep apnea, metabolic syndrome (Syndrome X), type IIdiabetes, diabetic complications including diabetic neuropathy,nephropathy, retinopathy, cataracts, heart failure, inflammation,thrombosis, congestive heart failure, and any other cardiovasculardisease related to obesity or an overweight condition. Moreover, whilenot wishing to be bound by theory it has also been determined thatoleuropein binds to all of the PPAR receptors α, δ, and γ and modulatetheir actions coordinately. Whether or not this is the mechanism bywhich oleuropein acts remains to be determined. Furthermore, with thediscovery that oleuropein selectively modulates all of the PPARreceptors, the potential for using high throughput assays to identifyother similar/analogous PAN-PPAR modulators pharmacologically usefuloleuropein-like compounds which modulate these receptors stimulatanouslyis feasible. Such compounds will be useful in the treatment ofPPAR-mediated diseases and conditions as well as any for whicholeuropein was previously considered to be useful. In addition,oleuropein may be used in diagnostic assays to monitor the progressionof disease or to monitor the effectiveness of therapy with agents thatare administered to patients suffering from obesity or an obesityrelated condition, or who are prone to development of such conditions.

Proliferator-Activated Receptors (PPARs)

The Proliferator-Activated Receptors (PPARs) are members of the nuclearreceptor superfamily, which upon binding to specific DNA responseelements and in response to ligand binding, result in the activation ofseveral genes. The PPARs contain a DNA-binding domain, a ligand-bindingdomain, and a flexible hinge connecting the two. The PPARs function asligand-regulated transcription factors that control the expression oftarget genes by binding to their responsive DNA sequence (DNA responseelements or PPREs) as heterodimers with the retinoid X receptor (RXR).The target genes encode enzymes involved in lipid metabolism anddifferentiation of adipocytes. The PPAR receptor experiences aconformational change upon ligand binding, which results in activationof gene transcription. The term “nuclear receptor” mainly refers tofactors which control transcription of a target gene by binding upstreamfrom the target gene promoter in a nuclear receptor ligand-dependentfashion. However, some nuclear receptors lack nuclear receptor ligands.Consequently, even nuclear receptors which lack nuclear receptor ligandsare called “nuclear receptors” if their structural and functionalhomology places them in the nuclear receptor gene superfamily. Nuclearreceptors include estrogen receptors (ER), vitamin D receptors (VDR),peroxisome proliferator-activated receptors (PPAR), liver X receptors(LXR), retinoic acid receptors (RAR), retinoid X receptors (RXR),androgen receptors (AR), glucocorticoid receptors (GR), farnesoid xreceptors (FXR), mineralcorticoid receptors (MR) and the like forexample, but are not limited by these.

There are three known subtypes of PPARs. They include the subtypes PPARalpha, PPAR gamma, and PPAR delta. Natural agonists of the three typesof PPARs include fatty acids, which implicates them as criticalregulators in metabolic pathways involving energy storage andutilization. Furthermore, this also suggests that the PPARs may bepotential targets for development of therapeutics against disorders suchas obesity, and obesity-related disorders, including diabetes anddyslipidemia (Kliewer, et al., Recent Progress in Hormone Research,(2001), 56: 239-63).

PPARα is expressed predominantly in the liver and, to a lesser extent,in cardiac and skeletal muscle. It plays a crucial role in fatty acidoxidation in response to fasting, providing ketone bodies that serve asan energy source for peripheral tissues. PPARα knockout mice cannot meetenergy demands during fasting, and develop hypoglycemia, hyperlipidemia,and fatty liver (Kersten S, Seydoux J, Peters J M, Gonzalez F J,Desvergne B, and Wahli W, Peroxisome proliferator-activated receptoralpha mediates the adaptive response to fasting. J Clin Invest, 1999.103: p. 1489-98.). PPARα agonists include the fibrates, which are usedclinically to treat hypertriglyceridemia.

PPARγ has several critical roles. First, PPARγ is essential for fat celldifferentiation. PPARγ knockout mice lack adipose tissue (Barak Y,Nelson M C, Ong E S, Jones Y Z, Ruiz-Lozano P, Chien K R, Koder A, andEvans R M, PPAR gamma is required for placental, cardiac, and adiposetissue development. Mol Cell, 1999.4: p. 585-95.; Kubota N, Terauchi Y,Miki H, Tamemoto H, Yamauchi T, Komeda K, Satoh S, Nakano R, Ishii C,Sugiyama T, Eto K, Tsubamoto Y, Okuno A, Murakami K, Sekihara H,Hasegawa G, Naito M, Toyoshima Y, Tanaka S, Shiota K, Kitamura T, FujitaT, Ezaki O, Aizawa S, Kadowaki T, and et al., PPAR gamma mediateshigh-fat diet-induced adipocyte hypertrophy and insulin resistance. MolCell, 1999. 4: p. 597-609.; Rosen E D, Sarraf P, Troy A E, Bradwin G,Moore K, Milstone D S, Spiegelman B M, and Mortensen R M, PPAR gamma isrequired for the differentiation of adipose tissue in vivo and in vitro.Mol Cell, 1999. 4: p. 611-7.), and overexpression of PPARγ convertsnon-adipocytes into fat cells (Tontonoz P, Hu E, and Spiegelman B M,Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, alipid-activated transcription factor. Cell, 1994. 79: p. 1147-56.).Second, PPARγ acts as a fatty acid sensor that regulates whole-bodyglucose homeostasis. PPARγ activates genes involved in lipogenesis andlipid storage. It also modulates adipokine expression, increasingproduction of adiponectin, while it blocks expression of TNF-α andresistin. The net result of these changes increases insulin sensitivity.PPARγ is the molecular target for the thiazolidinedione class of drugsthat improve insulin sensitivity.

PPARδ, also known as PPARβ, is expressed ubiquitously, and regulatesexpression of genes involved in fatty acid catabolism and adaptivethermogenesis. Transgenic expression of PPARδ results in lean mice thatare resistant to obesity and hyperlipidemia, while PPARδ knockout miceshow reduced energy uncoupling, and are prone to obesity (Wang Y X, LeeC H, Tiep S, Yu R T, Ham J, Kang H, and Evans R M,Peroxisome-proliferator-activated receptor delta activates fatmetabolism to prevent obesity. Cell, 2003. 113: p. 159-70.).

PPARs may modulate vascular effects by affecting insulin resistance, andby expression of adipokines. In addition, PPARs may have direct effectson gene transcription in vascular tissues and endothelial cells.

The significance of these receptors in physiology and disease isevidenced by the fact that PPAR-γ and PPARα are respective moleculartargets for the insulin-sensitizing thiazolidinedione (TZD) andlipid-lowering fibrate drugs that total more than $5 billion in annualsales.

PPARδ agonist GW501516(2-methyl-4-(((4-methyl-2-(4-trifluoromethylphenyl)-1,3-thiazol-5-yl)-methyl)sulfanyl)phenoxy)aceticacid) and PPARδ, α, γ, pan agonist GW2433(2-(4-(3-(1-(2-(2-chloro-6-fluoro-phenyl)-ethyl)-3-(2,3-dichloro-phenyl)-ureido)-propyl)-phenoxy)-2-methyl-propionicacid) were shown to lower plasma triglyceride levels in obese monkeyswhile raising high-density lipoprotein levels, prompting the initiationof clinical trials to assess efficacy in hyperlipidemic patients. Themedical potential of PPAR δ agonist and PPARδ, α, γ, pan agonist isbelieved to exceed that of both PPARα and PPARγ single agonist or dualagonists.

The findings presented herein demonstrate that oleuropein interacts withPPARα, PPARδ and PPARγ, and as such, offers a novel application fortherapeutic pan modulation of these important transcription factors inthe treatment of obesity and related disorders.

PPAR gamma: Out of the three subtypes, PPAR gamma has been mostextensively studied. It is known to play an important role in theregulation of glucose and lipid homeostasis as well as in adipocytedifferentiation (Willson, et al., Journal of Medicinal Chemistry,(2000), 43: 527-550). The PPAR gamma protein is conserved across severalspecies including mice and humans. One of the first synthetic ligands ofPPAR gamma identified as agonists was a class of antidiabetic compoundsknown as thiazolidinediones (TZDs). The efficacy of individual TZDs inanti-diabetic therapy appears to correlate with their ability to bindand activate the PPAR gamma receptor (Auwerx, J., Diabetologia, (1999),42: 1033-1049). This class of compounds has also been shown to inducegene expression in adipocytes, which correlates with lowered glucoselevels (Willson, et al.). TZDs have also been shown to reduce lipid andinsulin levels. Known thiazolidinedionies include Rosiglitazone,Troglitazone, Pioglitazone, and MCC-555. Each of these compounds bindspreferentially to PPAR gamma over the other PPAR subtypes. Pioglitazoneand rosiglitazone are Food and Drug Administration approved drugs thatare currently sold for the treatment of Type II diabetes. Troglitazonewas also FDA approved for Type II diabetes, but has been withdrawn fromcommercial use due to the occurrence of undesirable side effects.

Although the advances made with the thiazolidinedione class ofantidiabetes agents is significant, there are unacceptable side effectsassociated with this class of drugs, which have limited their clinicaluse. Accordingly, there remains a need for potent, selective modulatorsof PPAR gamma, of which the activators of PPAR gamma will be useful forthe treatment of NIDDM and other disorders related to lipid metabolismand energy homeostasis. Still further, compounds that block PPAR gammaactivity would be useful for interfering with the maturation ofpreadipocytes into adipocytes and thus would be useful for the treatmentof obesity and obesity-related disorders associated with undesirableadipocyte maturation. In addition, the response of patients toparticular TZDs is variable, with about 20-30% of patients using thesecompounds being classified as non-responders. Accordingly, it is highlydesirable to identify compounds for treating diabetes, as well as fortreating obesity and other obesity-related disorders that are moretherapeutically effective with fewer side effects. There is also a needto develop more accurate methods for predicting whether a subject islikely to respond to a particular treatment as well as methods thatdetermine the extent to which a patient has responded to the treatment.

PPAR gamma has been shown to be expressed in an adipose tissue-specificmanner. Furthermore, it is induced early during the course ofdifferentiation of preadipocytes. Further studies have now demonstratedthat PPAR gamma not only plays a pivotal role in the adipogenicsignaling cascade, but also regulates the ob/leptin gene which isinvolved in regulating energy homeostasis. PPAR gamma also plays a rolein adipocyte differentiation, which has been shown to be a critical stepfor targeting therapeutics for treating obesity and obesity-relatedconditions, such as diabetes.

PPAR gamma ligands also have in vitro anticancer activity against a widevariety of neoplastic cells and in vivo anticancer effects andchemotherapeutic or chemopreventive effects have been seen in animalstudies. PPAR gamma ligands may slow the growth of cancer cells and mayinduce the partial differentiation of some cancer cells. Overall, muchliterature indicates that PPAR gamma ligands have antiproliferativeactivity and may be useful in the treatment of cancer, includingparticularly several common cancers, including those of the colon,prostate, and breast. (See, Koeffler H P Clin Cancer Res. 9(1):1-9(2003)).

Furthermore, there is evidence to suggest that PPAR (e.g., PPAR-gamma)acts by a number of mechanisms to influence the permeability of skin,inhibit the growth of epidermal cells, promote the terminaldifferentiation of epidermis, and regulate the inflammatory response ofskin. Accordingly, PPAR ligands may be useful in the modulation of skinconditions characterized by hyperproliferation, inflammatory infiltratesand abnormal differentiation (e.g., psoriasis), including inflammatoryskin diseases (e.g. atopic dermatitis), proliferative skin diseases,acne vulgaris, protease inhibitor associated lipodystrophia and woundhealing. (See, Kuenzli S, et al., Br J. Dermatol. 149(2):229-36 (2003)).

Additional studies suggest that PPAR gamma plays a role in thepathophysiology of senile osteoporosis. For example, adipogenesis inbone marrow increases with aging. Mesenchymal stem cells expressing asubtype of this receptor (PPAR gamma 2) differentiate into adipocytes.Appropriate modulation of this receptor may promote mesenchymal stemcell differentiation into osteoblasts. Furthermore, activators of PPARalpha, delta, and gamma have been reported to induce alkalinephosphatase activity and bone matrix calcification. Accordingly,pharmacological use of PPAR activators should promote bone mineraldensity by modulating osteoblastic maturation. (See, Duque G, Drug NewsPerspect. 16(6):341-6 (2003); Jackson S M, et al., FEBS Lett.471(1):119-24 (2000). Based on the studies presented herein, anotheraspect of the present invention is the use of oleuropein, or an analogueor derivative thereof for the treatment of osteoporosis, or otherdiseases or conditions associated with bone loss.

The anti-proliferative and anti-inflammatory effects of PPAR gamma areobserved in glial cells and lymphocytes. It has been postulated thatactivation of such cells may be involved in the pathophysiology ofneurological diseases associated with brain inflammation (e.g,Alzheimer's disease and multiple sclerosis). Studies indicate that PPARgamma modulators would be therapeutically useful in such diseases. (See,Feinstein D L, Diabetes Technol Ther. 5(1):67-73 (2003)). Accordingly,the present invention provides for a method of treating inflammatorydiseases or conditions comprising administering a PPAR gamma modulatorto a subject in need of such therapy. In one particular embodiment, themethod provides for treating neurological or neurodegenerative diseasesor conditions caused in part by the presence or influx of inflammatorycells, such as for example, multiple sclerosis, stroke or Alzheimer'sdisease. The use of a PPAR modulator for treating a nervous systeminjury is also contemplated, for example, a spinal cord injury ortraumatic brain injury. In one particular embodiment, the PPAR modulatoris a PPAR gamma agonist.

Based on the studies done with the PPAR family of receptors in variousdisease conditions, it is possible that compounds that interact with thevarious PPAR receptors may be useful in treating these various diseases.Depending on the biological environment (e.g., cell type, pathologicalcondition of the host, etc.), these compounds can activate or block theactions of PPARgamma. By activating the PPAR gamma receptor, thecompounds find use as therapeutic agents capable of modulatingconditions mediated by the PPAR gamma receptor. As noted above, anexample of such a condition is non-insulin dependent diabetes mellitus(NIDDM). Additionally, the compounds may be useful for the preventionand treatment of complications of diabetes (e.g., neuropathy,retinopathy, glomerulosclerosis, and cardiovascular disorders), andtreating hyperlipidemia. Still further, the compounds may be useful forthe modulation of inflammatory conditions which most recently have beenfound to be controlled by PPAR gamma (see, Ricote, et al., Nature,391:79-82 (1998) and Jiang, et al., Nature, 391:82-86 (1998). Examplesof inflammatory conditions include asthma, rheumatoid arthritis andatherosclerosis. Such compounds may also be useful in the treatment ofother skin diseases such as acne, atopic dermatitis, psoriasis,photodermatitis, eczema, and seborrhea.

It has also been described in WO 96/33724 that compounds selective forPPAR gamma receptors, such as prostaglandin-J2 or -D2, may bepotentially active agents for the treatment of obesity and diabetes.Compounds that act via antagonism of PPAR gamma may be useful fortreating obesity, hypertension, hyperlipidemia, hypercholesterolemia,hyperlipoproteinemia, and metabolic disorders.

Compounds which are PPAR gamma agonists or activators by virtue of theiranti-inflammatory effects can have neuroprotective effects and find usein the treatment of brain inflammatory conditions such as Alzheimer'sdisease and multiple sclerosis. PPAR alpha, PPAR delta and their ligandsare important for lipid oxidation and energy uncoupling. They increasefat consumption and decrease fat accumulation, thus they can have weightreduction and anti-obesity effects. A ligand such as oleuropein with thecapability to bind all of the three PPARs has the potential to act as aPAN modulator. These are discussed below.

PPAR Gamma

Owing to their ability to induce gene expression in adipocytes and toenhance adipocyte differentiation, TZDs induce weight gain in oftenalready obese patients. For this reason, efforts are being made toidentify new partial agonists or antagonists for PPARγ in an attempt tocombine their anti-diabetic and anti-obesity effects.

Combined PPARα/PPARγ Agonists

The effects of TZDs on the lipid profile in diabetic patients are notoptimal. Given the favorable effect of PPAR activators on plasmalipoprotein metabolism, combined activation of PPARα and PPARγ couldlead to a complementary and synergistic action on lipid metabolism,insulin sensitivity and inflammation control. Dual activation of PPARαand γ could, in theory, also limit the occurrence of side effectsassociated with TZD therapy, such as edema and body-weight gain,although this has not been observed so far in clinical trials withcoagonists. Thus, combined PPARα/γ activation has recently emerged as anintriguing concept and spawned the development of co-agonists.

PPAR-Delta and PPARαδγ Pan Agonist or Modulator

Because of its ubiquitous expression and the paucity of selectiveligands, PPAR-delta is the least understood PPAR subtype. Nevertheless,early PPAR-6-selective agonists were found to elevate HDL-C levels indiabetic mice, an observation that indicated that PPARδ ligands mighthave beneficial effects on dyslipidemia (Eur J Pharmacol. 2006 Apr. 24;536(1-2):182-91. Epub 2006 Feb. 28). Subsequently, the potent PPARδagonist GW501516 was shown to increase HDL-C while decreasing elevatedTG and insulin levels in obese rhesus monkeys. GW501516 also attenuatesweight gain and insulin resistance in mice fed high-fat diets byincreasing the expression in skeletal muscle of genes that promote lipidcatabolism and mitochondrial uncoupling, thereby increasing β-oxidationof fatty acids in skeletal muscle (Curr Opin Investig Drugs. 2006 April;7(4):360-70).

Expected Results of PPAR Pan Modulator Such asOLE/Oleuropein/Hydroxytyrosol) on Metabolic Endpoints

Our discovery that Oleuropein is able to act as a PPAR pan agonist thattargets all of the three isoforms of PPARs, PPARα, PPARδ and PPARγoffers a new class of orally available potential novel drug. A compoundtargeting all of these PPAR subtypes could provide a combination oftriglyceride, LDL and glucose lowering activities, coupled withincreases in insulin sensitivity, HDL and reverse cholesterol transport(see Table 1). Its antihyperglycemic, lipid-modulating,insulin-sensitizing activities could be used in the treatment of avariety of metabolic and cardiovascular diseases, including Type IIdiabetes, impaired glucose tolerance, dyslipidemia, hypertension,metabolic syndrome X, asthma and HIV/HAART associated lipodystrophys.Furthermore, synergies of such a combination may enable lower dosing andconsequently mitigate side effects and toxicities observed with currenttherapies. Although treatment options for Type II diabetes areavailable, their usefulness is significantly limited due to theirfailure to ameliorate concurrent hyperglycemia and hyperlipidemia (e.g.triglycerides, LDL-cholesterol) or to raise HDL, in addition to theirside effects.

TABLE 1 Cardiovascular Disease PPAR Pan-Agonist Fact Sheet and ExpectedResults of OLE/Oleuropein/Hydroxytyrosol on Metabolic Endpoints TargetGlucose Insulin TG FAA LDL HDL Limitations PPARα No effect No effect ↓ ↓↓ ↑ Ineffective on glucose and insulin sensitivity PPARδ No effect ↑ ↓↓↓ ↓ ↑ Less validation for PPARδ PPARγ* ↓↓ ↑ ↓ ↓ ↑↑ No effect Edema,weight gain, anemia, LDL/cholesterol↑, need to monitor liver functionPPARαγ* ↓ ↑ ↓ ↓ ↓ ↑ Edema, weight gain, anemia, LDL/cholesterol↑, needto monitor liver function PPARαδγ** ↓↓ ↑↑ ↓↓↓ ↓↓ ↓↓ ↑↑↑ Less validationfor PPARδ Abbreviations: Insulin S, insulin sensitivity; TG,triglycerides; FFA, free fatty acids; LDL, low-density lipoproteincholesterol; HDL, high-density lipoprotein *Reported clinical effects inobese, rhesus monkeys and in patients Edema, weight gain, anemia, ↑ LDLcholesterol, requirement to monitor liver **Predicted effect ofoleuropein and derivativesUse of Oleuroein and/or Hydroxytyrosol for Inhibiting Viral Infectivity

The work presented herein also demonstrates that oleuropein (Ole) andhydroxytyrosol (HT) are a unique class of HIV-1 inhibitors from oliveleaf extracts, which are effective against viral fusion and integration.Molecular docking simulation was used to study the interactions of Oleand HT with viral targets. It was determined that Ole and HT bind to theconserved hydrophobic pocket on the surface of the HIV-gp41 fusiondomain by hydrogen bonds with Q577 and hydrophobic interactions withI573, G 572, and L 568 on gp41 N-terminal heptad repeat peptide N36,interfering with formation of the gp41 fusion-active core. To test andconfirm modeling predications, the effect of Ole and HT on HIV-1 fusioncomplex formation was studied using native polyacrylamide gelelectrophoresis and circular dichroism spectroscopy. Ole and HT exhibitdose dependent inhibition on HIV-1 fusion core formation with EC₅₀s of66-58 μM, with no detectable toxicity.

At present, 29 drugs are licensed by the FDA for the treatment of HIV-1infection in the United States [S. M. Hammer, Clinical practice.Management of newly diagnosed HIV infection, N Engl J Med 353 (2005)1702-1710]. These agents can be classified according to their mechanismof action: reverse transcriptase inhibitors (RTIs) (nucleoside, NRTIsand non-nucleoside, NNRTIs), protease inhibitors (PIs), fusioninhibitors, and multi-class combination products (MCCP). The combinationof RTIs and P is, commonly known as Highly Active Antiretroviral Therapy(HAART) [S. M. Hammer, Clinical practice. Management of newly diagnosedHIV infection, N Engl J Med 353 (2005) 1702-1710, J. Cohen, Therapies.Confronting the limits of success, Science 296 (2002) 2320-2324], hassignificantly reduced morbidity and mortality, transforming HIV/AIDSinto a manageable chronic illness. Although HAART can favorablyinfluence disease progression, it does not cure HIV infection. Antiviraltherapy must be maintained long-term, and serious chronic toxicity,therapy fatigue, and drug resistance have become major issues.

New therapeutic approaches include the fusion inhibitor Fuzeon (T-20,enfuvirtide), the non-peptidic PI Tipranavir, the new PI darunavir andthe recently approved MCCP Atripla. However, existing experience withHIV-1 highlights the need to use multiple effective agents incombination for maximal and durable effect. Thus, the search for novelanti-HIV agents continues to be of great significance, especially thosecapable of affecting multiple stages of the viral life cycle.

We previously reported that olive leaf extract is potent against HIV-1[S. Lee-Huang, L. Zhang, P. L. Huang, and Y. T. Chang, Anti-HIV activityof olive leaf extract (OLE) and modulation of host cell gene expressionby HIV-1 infection and OLE treatment, Biochem Biophys Res Commun 307(2003) 1029-1037]. Subsequent studies as described herein demonstratethat the anti-HIV properties of oleuropein (Ole) and its mainmetabolite, hydroxytyrosol (HT) are the key anti-HIV components of OliveLeaf Extract. They are active against multiple stages of the HIV-1 lifecycle, inhibiting cell-to-cell HIV-1 transmission and viral core antigenp24 production. Molecular docking simulations indicate that Ole and HTinteract with the conserved hydrophobic pocket on the surface of thecentral trimeric coiled-coil of HIV-gp41 fusion complex, the six helicalbundle (6HB), and the catalytic core domain (CCD) of HIV-1 integraseactive site.

Molecular modeling and functional confirmation of Ole and HT binding toHIV-1 integrase was also studied and the results summarized herein.Docking simulations identified two binding regions for Ole within theintegrase active site. Region I encompasses the conserved D64-D 116-E152 motif, while region II involves the flexible loop region formed byamino acid residues 140-149. HT, on the other hand, binds to region II.Both Ole and HT exhibit favorable interactions with important amino acidresidues through strong H-bonding and van der Waals contacts, predictingintegrase inhibition. To test and confirm modeling predictions, weexamined the effect of Ole and HT on HIV-1 integrase activitiesincluding 3′-processing, strand transfer and disintegration. Ole and HTexhibit dose-dependent inhibition on all three activities, with EC₅₀s inthe nM range. These studies demonstrate that molecular modeling oftarget-ligand interaction coupled with structural-activity analysisshould facilitate the design and identification of innovative integraseinhibitors and other therapeutics.

HIV-1 integrase is one of three viral enzymes required for viralreplication, along with RT and protease [P. O. Brown, Retroviruses, inCoffin, J., Hughes, S., and Varmus, H., (Eds.) Cold Spring Harbor Press,Cold Spring Harbor, 1998, 161-203; T. K. Chiu, and D. R. Davies,Structure and function of HIV-1 integrase, Curr Top Med Chem 4 (2004)965-977; K. Zhu, C. Dobard, and S. A. Chow, Requirement for integraseduring reverse transcription of human immunodeficiency virus type I andthe effect of cysteine mutations of integrase on its interactions withreverse transcriptase, J Virol 78 (2004) 5045-5055]. Integration ofHIV-1 cDNA into the host chromosome is essential for stable maintenanceof the viral genome, efficient viral gene expression and productiveinfection. Thus, viral integrase is a critical target for anti-HIVtherapy [Y. Pommier, A. A. Johnson, and C. Marchand, Integraseinhibitors to treat HIV/AIDS, Nat Rev Drug Discov 4 (2005) 236-248; P.A. Sherman, and J. A. Fyfe, Human immunodeficiency virus integrationprotein expressed in Escherichia coli possesses selective DNA cleavingactivity, Proc Natl Acad Sci USA 87 (1990) 5119-5123]. The first stepleading to viral DNA integration is the binding of viral integrase toHIV long terminal repeat (LTR) sequences. This is followed by threesequential reactions: 1) 3′-processing, the removal of two nucleotides,GT, from the 3′-end of HIV-LTR, 2) strand transfer, a concertedcleavage-ligation reaction, in which the integrase makes a staggered cutin the target DNA and ligates the recessed 3′ ends of the viral DNA tothe 5′ ends of the target DNA, and 3) gap repair, removal of the twounpaired nucleotides at the 5′ end of the viral DNA and repair of thegap between the viral and the target DNA. In the presence of a DNAsubstrate that mimics the product of viral integration, integrase cancatalyze the reversal of strand transfer, known as “disintegration” [S.A. Chow, K. A. Vincent, V. Ellison, and P. O. Brown, Reversal ofintegration and DNA splicing mediated by integrase of humanimmunodeficiency virus, Science 255 (1992) 723-726 S. A. Chow, K. A.Vincent, V. Ellison, and P. O. Brown, Reversal of integration and DNAsplicing mediated by integrase of human immunodeficiency virus, Science255 (1992) 723-726], in which viral DNA is released and the target DNAis sealed.

Therapeutic and Prophylactic Compositions and their Use

Candidates for therapy with the agents identified by the methodsdescribed herein are patients either suffering from, or at risk fordevelopment of obesity, or an obesity related disease, disorder orcondition, including diabetes, dyslipidemia, hypertension andcardiovascular diseases, asthma to name a few. Other obesity relateddisorders have been described previously. In addition, patients who areobese or are prone to developing obesity or an obesity related disorderbased on a genetic predisposition are also considered to be candidatesfor therapy using oleuropein or a compound having activity analogous tooleuropein. Furthermore, patients who have limited mobility or patientswho are bed-ridden due to a surgical procedure or illness and are notcapable of exercise and are thus prone to accumulation of body fat maybe candidates for therapy with the agents identified by the methodsdescribed. In addition, the present invention contemplates the use ofoleuropein, hydroxytyrosol, and their derivatives, analogues or mimicsthereof, for inhibiting the growth and/or infectivity of HIV-1 by virtueof the effects of oleuroein and hydroxytyrosol on inhibition of viralfusion and/or by their effects on the viral integrase.

The invention provides methods of treatment comprising administering toa subject an effective amount of an agent of the invention. In apreferred aspect, the compound is substantially purified (e.g.,substantially free from substances that limit its effect or produceundesired side-effects). The subject is preferably an animal, includingbut not limited to animals such as monkeys, cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human. In one specific embodiment, a non-human mammal is thesubject. In another specific embodiment, a human mammal is the subject.Accordingly, the agents identified by the methods described herein maybe formulated as pharmaceutical compositions to be used for prophylaxisor therapeutic use to treat these patients. Moreover, the agents of theinvention may be useful for administering to non-human mammals to aid inthe build-up of lean muscle which may then prove highly beneficial forthe meat industry.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, or microcapsules. Methods of introduction can be enteralor parenteral and include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, topical and oral routes. The compounds may be administered byany convenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compositions of the invention into the central nervoussystem by any suitable route, including intraventricular and intrathecalinjection; intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir, such asan Ommaya reservoir. Pulmonary administration can also be employed,e.g., by use of an inhaler or nebulizer, and formulation with anaerosolizing agent. In a specific embodiment, it may be desirable toadminister the pharmaceutical compositions of the invention locally tothe area in need of treatment.

Such compositions comprise a therapeutically effective amount of anagent, and a pharmaceutically acceptable carrier. In a particularembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the subject. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lidocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects (a)approval by the agency of manufacture, use or sale for humanadministration, (b) directions for use, or both.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved, for example, and not by way oflimitation, by local infusion during surgery, by topical application, byinjection, by means of a catheter, or by means of an implant, saidimplant being of a porous, non-porous, or gelatinous material, includingmembranes, such as sialastic membranes, or fibers or co-polymers such asElvax (see Ruan et al, (1992), Proc Natl Acad Sci USA, 89:10872-10876).In one embodiment, administration can be by direct injection by aerosolinhaler.

In another embodiment, the compound can be delivered in a vesicle, inparticular a liposome (see Langer (1990) Science 249:1527-1533; Treat etal., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

In yet another embodiment, the compound can be delivered in a controlledrelease system. In one embodiment, a pump may be used (see Langer,supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al.(1980) Surgery 88:507; Saudek et al. (1989) N. Engl. J. Med. 321:574).In another embodiment, polymeric materials can be used (see MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug ProductDesign and Performance, Smolen and Ball (eds.), Wiley, New York (1984);Ranger and Peppas, J. (1983) Macromol. Sci. Rev. Macromol. Chem. 23:61;see also Levy et al. (1985) Science 228:190; During et al. (1989) Ann.Neurol. 25:351; Howard et al. (1989) J. Neurosurg. 71:105). In yetanother embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the airways, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release (1984) supra, vol. 2, pp. 115-138).Other suitable controlled release systems are discussed in the review byLanger (1990) Science 249:1527-1533.

Effective Doses

Toxicity and therapeutic efficacy of compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage tounaffected cells and, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can beused in formulating a dose range for use in humans. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage can varywithin this range depending upon the dosage form employed and the routeof administration utilized. For any compound used in the method of theinvention, the therapeutically effective dose can be estimated initiallyfrom cell culture assays. A dose can be formulated in animal models toachieve a circulating plasma concentration range that includes the IC₅₀(i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to optimize efficacious doses for administrationto humans. Plasma levels can be measured by any technique known in theart, for example, by high performance liquid chromatography.

In addition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each subject's circumstances.Normal dose ranges used for particular therapeutic agents employed forspecific diseases can be found in the Physicians' Desk Reference,54^(th) Edition (2000).

While the subject is being treated, the health of the patient may bemonitored by measuring one or more relevant indices at predeterminedtimes during a 24-hour period. For example, lipid levels may bemonitored during the course of therapy using standard procedures knownto those skilled in the art, in order to monitor the effectiveness oftherapy. Alternatively, for patients suffering from HIV, blood samplesmay be obtained during the course of therapy to monitor levels of viralnucleic acid using standard PCR procedures known to those skilled in theart. Treatment, including supplement, amounts, times of administrationand formulation, may be optimized according to the results of suchmonitoring. The patient may be periodically reevaluated to determine theextent of improvement by measuring the same parameters, the first suchreevaluation typically occurring at the end of four weeks from the onsetof therapy, and subsequent reevaluations occurring every four to eightweeks during therapy and then every three months thereafter. Therapy maycontinue for several months or even years, with a minimum of one monthbeing a typical length of therapy for humans. Adjustments to theamount(s) of agent administered and possibly to the time ofadministration may be made based on these reevaluations.

Treatment may be initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage may be increased bysmall increments until the optimum therapeutic effect is attained.

The combined use of several compounds of the present invention, oralternatively other therapeutic agents, may reduce the required dosagefor any individual component because the onset and duration of effect ofthe different components may be complimentary. In such combined therapy,the different active agents may be delivered together or separately, andsimultaneously or at different times within the day.

The invention includes use of any modifications or equivalents of theabove agents which do not exhibit a significantly reduced activity. Thestatements of effect and use contained herein are therefore to beconstrued accordingly, with such uses and effects employing modified orequivalent gene products being part of the invention.

Oleuropein is a phenolic secoiridoid glycoside with the structure asshown in the figure below. It is an organic molecule that consists of adihydroxy phenol moiety, a secoiridoid moiety and a glucose moiety. Theglucose moiety can be removed by β-glycosidase to yield oleuropeinaglycone. Oleuropein aglycone can than be hydrolyzed into hydroxytyrosoland elenolic acid. These are the metabolites of oleuropein and they areall physiological active (See FIG. 10).

The present agents that prevent fat accumulation, or that modulatedifferentiation, de-differentiation and trans-differentiation ofadipocytes or that have an effect on the expression of adipogenic,lipogenic and lipolytic genes and gene products can be used as the soleactive agents, or can be used in combination with other activeingredients

Combination Therapy

The compounds of the invention may be combined with another therapeuticagent that is useful in the treatment of obesity or disorders associatedwith the development and progression of obesity and obesity relateddisorders, such as, diabetes, atherosclerosis, hypertension,hyperlipidaemias, dyslipidaemias, and cardiovascular disease. Thecompounds of the invention may be combined with another therapeuticagent that decreases the ratio of LDL:HDL or an agent that causes adecrease in circulating levels of LDL-cholesterol. In patients withdiabetes mellitus the compounds of the invention may also be combinedwith therapeutic agents used to lower blood sugar or to treatcomplications associated with diabetes, including nephropathy,neuropathy, retinopathy, cardiovascular disease, stroke, or any othercomplication associated with diabetes.

Accordingly, the compounds of the invention may be used alongside othertherapies for the treatment of metabolic syndrome or type 2 diabetes andits associated complications, these include biguanide drugs, for examplemetformin, phenformin and buformin, insulin (synthetic insulinanalogues, amylin) and oral antihyperglycemics (these are divided intoprandial glucose regulators and alpha-glucosidase inhibitors). Anexample of an alpha-glucosidase inhibitor is acarbose or voglibose ormiglitol. An example of a prandial glucose regulator is repaglinide ornateglinide. Alternatively, oleuropein and/or hydroxytyrosol may be usedin combination with other anti-virals known in the art to be effectiveagainst HIV.

In another aspect of the invention, the agents of the invention, e.g.oleuropein or an analogue or derivative thereof, or a pharmaceuticallyacceptable salt, solvate, solvate of such a salt or a prodrug thereof,may be administered in association with another PPAR modulating agent.PPAR modulating agents include but are not limited to a PPAR alpha,delta and/or gamma agonist, or pharmaceutically acceptable salts,solvates, solvates of such salts or prodrugs thereof. Suitable PPARalpha, delta and/or gamma agonists, pharmaceutically acceptable salts,solvates, solvates of such salts or prodrugs thereof are well known inthe art. These include the compounds described in WO 01/12187, WO01/12612, WO 99/62870, WO 99/62872, WO 99/62871, WO 98/57941, WO01/40170, J Med Chem, 1996, 39, 665, Expert Opinion on TherapeuticPatents, 10 (5), 623-634 and J Med Chem, 2000, 43, 527, which are allincorporated herein by reference in their entireties. Particularly aPPAR alpha and/or gamma agonist refers to NN622/Ragaglitazar, BMS298585, WY-14643, clofibrate, fenofibrate, bezafibrate, gemfibrozil andciprofibrate; GW 9578, ciglitazone, troglitazone, pioglitazone,rosiglitazone, eglitazone, proglitazone, BRL-49634, KRP-297, JIT-501, SB213068, GW 1929, GW 7845, GW 0207, L-796449, L-165041, GW 2433 and aPPAR alpha and/or gamma agonist such as(S)-2-ethoxy-3-[4-(2-{4-methanesulphonyloxyphenyl}ethox-y)-phenyl]propanoicacid and pharmaceutically acceptable salts thereof.

In addition the combination of the invention may be used in conjunctionwith a sulfonylurea for example: glimepiride, glibenclamide (glyburide),gliclazide, glipizide, gliquidone, chloropropamide, tolbutamide,acetohexamide, glycopyramide, carbutamide, glibonuride, glisoxepid,glybuthiazole, glibuzole, glyhexamide, glymidine, glypinamide,phenbutamide, tolcylamide and tolazamide. Preferably the sulfonylurea isglimepiride or glibenclamide (glyburide). More preferably thesulfonylurea is glimepiride. Therefore the present invention includesadministration of a compound of the present invention in conjunctionwith one, two or more existing therapies described in this paragraph.The doses of the other existing therapies for the treatment of obesityor an obesity related disorder, such as, type 2 diabetes and itsassociated complications will be those known in the art and approved foruse by regulatory bodies for example the FDA and may be found in theOrange Book published by the FDA. Alternatively smaller doses may beused as a result of the benefits derived from the combination. Thepresent invention also includes a compound of the present invention incombination with a cholesterol-lowering agent. The cholesterol-loweringagents referred to in this application include but are not limited toinhibitors of HMG-CoA reductase (3-hydroxy-3-methylglutaryl coenzyme Areductase). Suitably the HMG-CoA reductase inhibitor is a statinselected from the group consisting of atorvastatin, bervastatin,cerivastatin, dalvastatin, fluvastatin, itavastatin, lovastatin,mevastatin, nicostatin, nivastatin, pravastatin and simvastatin, or apharmaceutically acceptable salt, especially sodium or calcium, or asolvate thereof, or a solvate of such a salt. A particular statin isatorvastatin, or a pharmaceutically acceptable salt, solvate, solvate ofsuch a salt or a prodrug thereof. More particular statins arerosuvastatin and atorvastatin calcium salts.

In the present application, the term “cholesterol-lowering agent” alsoincludes chemical modifications of the HMG-CoA reductase inhibitors,such as esters, prodrugs and metabolites, whether active or inactive.

Diabetes mellitus is a syndrome resulting from the interaction ofhereditary and environmental factors; it is characterized bydisturbances in insulin secretion and other metabolic and vascularabnormalities, i.e. an elevated concentration of glucose in the blood,non-specific accelerated arteriosclerosis, neuropathy and thickening ofthe capillary basal lamina caused by a degeneration of the kidney andthe retina.

According to a modern classification, the diabetes is divided into twomain categories:

Insulin-dependent diabetes mellitus (also known as Type I diabetes);patients with this type of diabetes literally depend on insulin toprevent ketoacidosis and death. As far as the endogenous insulinsecretion is concerned, patients with Type I diabetes mellitus exhibitinsulinopenia.

Noninsulin-dependent diabetes mellitus (also known as Type 2 diabetes);patients with this type of diabetes do not need insulin to live: theycan decide whether using it or not to control the symptoms of thediabetes. As far as the endogenous insulin secretion is concerned,patients with Type 2 diabetes can be further classified into two groups.In the first group, insulin levels are either normal or lower thannormal; in the second group, insulin values are higher than normal andpatients exhibit insulin resistance.

Accordingly, given the data presented herein, oleuropein may be usefulas a stand-alone agent for the treatment of obesity, or for preventingfat accumulation in individuals prone to obesity. However, it is also tobe understood that oleuropein may be used as adjunct therapy with otherknown agents used to treat obesity or obesity related disorders.

Monitoring a Patient's Response to the Therapeutic

Monitoring a patient's response to the therapeutic is usually done bymeasuring metabolic endpoints including plasma levels of glucose,insulin, TG, FAA, LDL, HDL in addition to adipogenic, lipogenic andlipolytic genes and gene products (proteins and/or enzymes). These areusually evaluated from whole blood, blood cells, plasma or serum of thepatient's blood by specific bioassays. These assays are routinely usedin clinical analysis of patient's blood samples. The modulation of thePPARs (genes or proteins), adipogenic, lipogenic and lipolytic genes andgene products, cytokines, hormones (insulin etc), adipokines as well asother genes and gene products involved in the targeted regulatorypathways can be determined from peripheral blood mononuclear cells(PBMC) purified from the blood sample using RT-PCR, cDNA microarrayblots for specific genes and ELISA or proteomics for gene products(proteins). Specific enzyme assays can be used for monitoring enzymaticactivities. An example of the metabolic variables to be measured in aproposed clinical trial is presented in Table 2 below. However, inaddition to the variables described below, other measurements mayinclude, but not be limited to, determination of the specific levels ofcytokine and adipokines such as IL-6, resistin, MCP-1, PAI-1,adiponection or leptin.

TABLE 2 Metabolic Variables at Baseline and the Changes Observed after,for example, 3 months, 6 Months and 12 Months of Treatment withOleuropein, and derivatives (including but not limit to its metabolites)for diabetics/obesity/cardiovascular disease Variable Mean Change after3, 6, or 12 Months or Baseline other defined times Placebo OLE PlaceboOLE Group Group Group Group P Metabolic characteristics (n = xx) (n =xx) (n = xx) (n = xx) Value† Ratio of glucose disposal rate to meanserum insulin level at steady state Fasting glucose level, mmol/L(mg/dL) 2-h glucose level, mmol/L (mg/dL) Fasting insulin level, pmol/LInsulin AUC level, pmol/L × 10⁴ (120 min) Total cholesterol level,mmol/L (mg/dL) LDL cholesterol level, mmol/L (mg/dL) HDL cholesterollevel, mmol/L (mg/dL) Triglyceride level, mmol/L (mg/dL) Adiponectinlevel, μg/mL Free fatty acid level, mmol/L TNFα, IL-6, adipokines PPARα,PPARδ, PPARγ Adipogenic genes and gene products Lipogenic genes and geneproducts Lipolytic genes and gene products Safety variables Hemoglobinlevel, g/L ALT level, U/L * All values are means ± SD. ALT = alanineaminotransferase; AUC = area under the curve; HDL = high-densitylipoprotein; LDL = low-density lipoprotein. †P values for mean changefrom baseline represent between-group effect from analysis of covariancewith baseline value as covariate. For treating AIDS relatedlipodystrophy one also measure CD⁺ counts and Log HIV RNA level,copies/mL

The present invention further provides for methods of diagnosing apatient's response to treatment with oleuropein or an analogue orderivative thereof. Furthermore, the present invention providesprognostic methods for evaluating the progression of treatment witholeuropein or an analogue or derivative thereof. The invention providesa array of genes and gene products identified as being modulatedfollowing treatment with oleuropein or an analogue or derivative thereofand which may be used to monitor a therapeutic response to treatmentwith oleuropein or an analogue or derivative thereof. The genes and geneproducts, which are up- or down-regulated in response to treatment witholeuropein may be used diagnostically and prognostically for treatmentof a PPAR associated disease, such as obesity or an obesity relateddisorder or disease such as Type II diabetes, with a PPAR ligand. Thesegenes or gene products (proteins or enzymes) can be monitored from thepatient's blood (whole blood cells, serum or plasma) by enzyme assay,ELISA (antibody), RT-PCR, microarray, and/or proteomics techniques.Exemplary diagnostic tools and assays are set forth below, underfollowed by exemplary methods for conducting these assays.

Diagnostic Tools and Assays and Methods for Identifying NovelTherapeutics for Treating a Disease Associated with a PPAR Receptor

One aspect of the invention provides a means of determining whether asubject is responsive to treatment with oleuropein or an analogue orderivative thereof or a combination of oleuropein and a second agentused to treat obesity or an obesity related disorder, or of assessingthe final outcome of therapy with oleuropein or an analogue orderivative thereof or a combination of oleuropein and a second agentused to treat obesity or an obesity related disorder in a subject inneed of such therapy. Another aspect of the invention provides for theuse of methods for screening for novel therapeutics for treating adisease associated with a PPAR receptor. The methods described hereinare merely exemplary and are not meant to be limiting, and as such, itis to be recognized that one of skill in the art would be cognizant ofthe various other methodologies that may be used to determineeffectiveness of therapy with oleuropein or analogues or derivativesthereof, or to identify novel analogues or derivatives or metabolites ofoleuropein for use in treating obesity or obesity related disorders.

In one particular embodiment, the method comprises measuring any one ormore of the following clinical parameters: lipid profile, glucoselevels, insulin levels, or any one or more cytokines or adipokinespresent in the serum or plasma of a subject undergoing treatment witholeuropein, or an analogue or derivative or metabolite of oleuropein.Any one or more of these sensitive and reliable end-points may beeffective at determining whether oleuropein, or an analogue, or aderivative, or a metabolite thereof, or a combination of oleuropein oran analogue or derivative or metabolite thereof together with one ormore agents useful for treating obesity or an obesity related disorderhas proven to be effective in treating these patients suffering fromthese conditions.

In another particular embodiment, the effectiveness of therapy witholeuropein or hydroxytyrosol or an analogue or derivative thereof inpatients infected with HIV may be measured by standard procedures knownto those skilled in the art. For example, in the early stages ofinfection, HIV often causes no symptoms and the infection can bediagnosed only by testing a person's blood. Two tests are available todiagnose HIV infection—one that looks for the presence of antibodiesproduced by the body in response to HIV and the other that looks for thevirus itself. The first test is an (Enzyme Linked Immunosorbent Assay).an ELISA test and it is used to measure antibodies produced by thepatient against the virus. When the body is infected with HIV, one looksfor such antibodies in blood.

If antibodies are present, the test gives a positive result, but thismust be confirmed by another test called Western Blot orImmunofluoroscent Assay (IFA). All positive tests by ELISA may not beaccurate and hence Western Blot and repeated tests are necessary toconfirm a person's HIV status. A person infected with HIV is termedHIV—positive or seropositive.

Antibodies to HIV generally do not reach detectable levels in the bloodtill about three months after infection. This period, from the time ofinfection till the blood is tested positive for antibodies, is calledthe Window Period. Some times, the antibodies might take even six monthsto show up. Even if the tests are negative, during the Window Period,the amount of virus is very high in an infected person. Hence, if aperson is newly infected, the risk of transmission is higher.

If a person is highly likely to be infected with HIV and yet both thetests are negative, a doctor may suggest a repetition of the tests afterthree months or six months when the antibodies are more likely to havedeveloped.

The second test is called PCR (Polymerase Chain Reaction), which looksfor HIV itself in the blood. This test, which recognizes the presence ofthe virus' genetic material in the blood, can detect the virus within afew days of infection.

There are also tests like Radio Immuno Precipitation Assay (RIPA), aconfirmatory blood test that may be used when antibody levels aredifficult to detect or when Western Blot test results are uncertain.Other available tests are Rapid Latex Agglutination Assay, a simplified,inexpensive blood test that may prove useful in medically disadvantagedareas where there is a high prevalence of HIV infection, and p24 AntigenCapture Assay.

In another particular embodiment, the responsiveness of a subject totreatment with oleuropein or an analogue or derivative or metabolitethereof can be assessed by measuring body mass index (BMI) or bymonitoring the weight of the subject. In a particular embodiment, onewould expect to see an improvement in the BMI or a decrease in bodyweight or at least a stabilization in body mass (eg. no additionalweight gain over a given period of time).

In another particular embodiment, such a method comprises determiningthe levels of expression of one or more genes or gene products(proteins) which are modulated in a cell of the subject undergoingtreatment with oleuropein (or an oleuropein analogue, derivative, ormetabolite thereof) and comparing these levels of expression with thelevels of expression of the genes and gene products in a cell of asubject not treated with oleuropein (or an oleuropein analogue,derivative, or metabolite thereof), or of the same subject beforetreatment with oleuropein (or an oleuropein analogue, derivative, ormetabolite thereof), such that the modulation (either up ordown-regulation of the gene or gene product) of one or more genes isindicative that the subject is responsive to treatment with oleuropein(or with an analogue, derivative or metabolite thereof). In oneembodiment, the cell is obtained from a sample of whole blood, forexample, white blood cells, including lymphocytes, monocytes,neutrophils and the like, although other cells expressing these genesare also contemplated for analysis. In one embodiment, the genes may beany of the adipogenic genes or gene products selected from the groupconsisting of Peroxisome Proliferator-Activated Receptor γ2 (PPARγ2),lipoprotein lipase (LPL), and the adipocyte-selective fatty acid bindingprotein (the αP2 gene). In addition, other differentiated adipocytemarker genes include glycerophosphate dehydrogenase (GPDH), fatty acidsynthase, acetyl CoA carboxylase, malic enzyme, Glut 4, and the insulinreceptor (see Spiegelman et al. J. Biol. Chem. 268: 6823-6826, 1993,incorporated herein by reference). Preadipocytes also havecharacteristic marker genes, such as the cell surface antigen recognizedby the monoclonal antibody AD-3. Expression level changes of the variousisoforms of the C/EBP (CCAAT/enhancer-binding proteins) family oftranscription factors may also indicate different stages of adipogenesis(see Yu and Hausman, Exp Cell Res Dec. 15, 1998; 245(2): 343-9). Aperson of skill in the art will recognize that in certain diagnostic andprognostic assays, it will be sufficient to assess the level ofexpression of a single adipogenic, lipogenic or lipolytic gene as notedabove and that in others, the expression of two or more is preferred.For example, the level of expression of a gene or gene product (protein)may be determined by a method selected from, but not limited to, cDNAmicroarray, reverse transcription-polymerase chain reaction (RT-PCR),real time PCR and proteomics analysis. Other means such aselectrophoretic gel analysis, enzyme immunoassays (ELISA assays),Western blots, dotblot analysis, Northern blot analysis and in situhybridization may also be contemplated for use, although it is to beunderstood that the former assays that are noted (eg. microarrays,RT-PCR, real time PCR and proteomics analysis) provide a more sensitive,quantitative and reliable measurement of genes or gene products that aremodulated by oleuropein or analogues, derivatives or metabolitesthereof. Sequences of the genes or cDNA from which probes are made (ifneeded) for analysis may be obtained, e.g., from GenBank, other publicdatabases or publications, and are shown here for certain of theexemplary markers shown in Tables 3 and 4. Magnetic resonance imagingmay also be used for assessing the effect of oleuropein or analogues orderivatives or metabolites thereof on protein expression.

In another particular embodiment, novel candidate therapeutics (eg.oleuropein analogues or derivatives or metabolites thereof) may betested for activity by measuring their effect on adipocytedifferentiation, de-differentiation or trans-differentiation, asdescribed herein. The candidate therapeutics may be selected from thefollowing classes of compounds: proteins, peptides, peptidomimetics,antibodies, derivatives of fatty acids, nucleic acids, including DNA orRNA, antisense molecules or siRNA molecules, or other small organicmolecules, either synthetic or naturally derived. In some embodiments,the candidate therapeutics are selected from a library of compounds.These libraries may be generated using combinatorial synthetic methods.

Use of Microarrays for Determining Gene Expression Levels

Microarrays may also be used for determining gene expression levels andmay be prepared by methods known in the art, or they may be custom madeby companies, e.g., Affymetrix (Santa Clara, Calif.) (seewww.affymetrix.com). Numerous articles describe the different microarraytechnologies, (e.g., Shena, et al., Tibtech, (1998), 16: 301; Duggan, etal., Nat. Genet., (1999), 21:10; Bowtell, et al., Nat. Genet., (1999),21:25; Hughes, et al., Nat. Biotechn., (2001), 19:342). While many ofthe microarrays utilize nucleic acids and relevant probes for theanalysis of gene expression profiles, protein arrays, in particular,antibody arrays or glycosylation arrays also hold promise for studiesrelated to protein or glycoprotein expression from biological samples(see for example, RayBiotech, Inc. at www.raybiotech.com/product.htm,Panomics at www.panomics.com, Clontech Laboratories, inc. atwww.clontech.com, Procognia in Maidenhead, UK and Qiagen atwww.qiagen.com.

Samples for Analysis

While the efficacy of oleuropein or an analogue, derivative, ormetabolite thereof may be tested in a subject for its effect on, forexample, glucose levels, insulin levels, lipid profile, body mass indexor weight loss, it may also be of interest to assess its effects on themodulation of the genes or gene products listed in Table 4. While it maybe possible to look at the level of a particular gene in certaincellular samples (whole blood cells or peripheral blood mononuclearcells), a more particular method would involve the analysis of theprotein expression in these cell types or in the plasma or serum formthe subjects exposed or treated with oleuropein or an analogue orderivative thereof. For example, protein and nucleic acid prepared fromspecimens may be obtained from an individual to be tested using either“invasive” or “non-invasive” sampling means. A sampling means is said tobe “invasive” if it involves the collection of the biosamples fromwithin the skin or organs of an animal (including, especially, a human,a murine, an ovine, an equine, a bovine, a porcine, a canine, or afeline animal). Examples of invasive methods include needle biopsy,pleural aspiration, etc. Examples of such methods are discussed by Kim,C. H. et al., J. Virol., (1992), 66:3879-3882; Biswas, B. et al., AnnalsNY Acad. Sci., (1990), 590:582-583; Biswas, B., et al., J. Clin.Microbiol., (1991), 29:2228-2233. Extraction of adipose tissue fromindividuals used in some embodiments of this invention is well known tothose skilled in the art, for example as described by Lonnroth, et al.,Diabetes, (1983), 32980: 748-54.

In one embodiment the assays of the present invention will be performedon cells including but not limited to whole blood cells, or isolatedwhite blood cells from a mammal, or from adipocyte cultures propagatedfor laboratory purposes, 3T3-L1 adipocytes, cells of skeletal musclederived from a mammal, skeletal muscle cells propagated for laboratorypurposes, C2C12 myotube cells, or mesenchymal stem cells, etc. Primarycultures or cell lines can be used. Alternatively, embroyonic stem (ES)cells differentiated into adipocytes can be used, for example, asdescribed in Poliard, et al., Journal of Cell Biology, (1995), 130:1461-72. Appropriate cell lines that can be obtained for screeningpurposes are commercially available from the ATCC. In yet anotherembodiment, a sample of whole blood, blood plasma or serum is obtainedfor further analysis.

Other Methods for Determining Gene Expression Levels

In certain embodiments, it is sufficient to determine the expression ofone or only a few genes, as opposed to hundreds or thousands of genes.Although microarrays may be used in these embodiments, various othermethods of detection of gene expression are available.

For example, the modulation of gene expression can be performed using aRT-PCR or Real Time-PCR assay. Total RNA is extracted using proceduresknown to those skilled in the art and subjected to reverse transcriptionusing an RNA-directed DNA polymerase, such as reverse transcriptaseisolated from AMV, MoMuLV or recombinantly produced. The cDNAs producedcan be amplified in the presence of Taq polymerase and the amplificationmonitored in an appropriate apparatus in real time as a function of PCRcycle number under the appropriate conditions that yield measurablesignals, for example, in the presence of dyes that yield a particularabsorbance reading when bound to duplex DNA. The relative concentrationsof the mRNAs corresponding to chosen genes can be calculated from thecycle midpoints of their respective Real Time-PCR amplification curvesand compared between cells exposed to a candidate therapeutic relativeto a control cell in order to determine the increase or decrease in mRNAlevels in a quantitative fashion.

In addition, a method for high throughput analysis of gene expression isthe serial analysis of gene expression (SAGE) technique, first describedin Velculescu, et al., Science, (1995), 270, 484-487. Among theadvantages of SAGE is that it has the potential to provide detection ofall genes expressed in a given cell type, provides quantitativeinformation about the relative expression of such genes, permits readycomparison of gene expression of genes in two cells, and yields sequenceinformation that may be used to identify the detected genes. Thus far,SAGE methodology has proved itself to reliably detect expression ofregulated and nonregulated genes in a variety of cell types (Velculescu,et al., (1997), Cell, 88, 243-251; Zhang, et al., Science, (1997), 276,1268-1272 and Velculescu, et al., Nat Genet, (1999), 23, 387-388.Techniques for producing and probing nucleic acids are furtherdescribed, for example, in Sambrook, et al., Molecular Cloning: ALaboratory Manual (New York, Cold Spring Harbor Laboratory, 1989).

In other methods, the level of expression of a gene is detected bymeasuring the level of protein encoded by the gene. In the case ofpolypeptides which are secreted from cells, the level of expression ofthese polypeptides may be measured in biological fluids. While methodssuch as immunoprecipitation, ELISA, Western blot analysis, orimmunohistochemistry using an agent, e.g., an antibody, thatspecifically detects the protein encoded by the gene may becontemplated, other more sensitive and quantitative methods arepreferred, as described below. The invention is not limited to aparticular assay procedure, and therefore is intended to include bothhomogeneous and heterogeneous procedures. General techniques to be usedin performing the various immunoassays noted above are known to those ofordinary skill in the art.

Proteomics: Rationale for Use

While genomic profiling provides information about susceptibility todisease, proteomic profiling reflects snapshots of metabolic dynamics,reveals heterogeneous gene expression, identifies biologically relevantphenotypes and generates information on protein structure-functionrelationships in the severity and prognosis of a disease. Thus, resultsfrom proteomic studies should offer insight into the pathology ofobesity and effects by oleuropein therapy. Many forms of proteinalterations can be associated with pathophysiological changes andtherapeutic treatments. In addition to expression levels and patterns,these include alternative splicing, post-translational modifications,proteolytic processes, co-secretion and protein-protein interactions.Thus, the identification and quantification of proteins alone is notsufficient to understand functional interactions. Changes as small asthe addition of a single phosphate, cleavage of a leader peptide,amidation, or oxidation, can drastically alter the biological functionof a protein. Thus, it is important to detect these minute changes usingsensitive and accurate proteomic technology.

Sample Preparation

Serum or plasma proteins may be differentially expressed in response topathophysiological changes in obesity and related diseases and totherapeutic treatments of these disorders. Proteomic study of plasmaproteins in normal and obese patients demonstrated significantdifferences in protein patterns. Plasma will be prepared from bloodsamples and used in proteomic analyses.

Proteomics: The Use of 2DE, MS, MALDI-TOF, MS/MS, LC/MS, and SELDI-TOF

2-dimensional polyacrylamide gel electrophoresis (2DE) coupled to massspectrometry (MS) is currently the standard analysis in proteomics.Plasma samples may be subjected to 2DE (first dimension isoelecticfocusing, second dimension SDS-PAGE). Selected spots from 2DE may beextracted from the gels, digested with trypsin and subjected to MSanalysis to determine their identities (Aebersold, R. & Mann, M. Massspectrometry-based proteomics. Nature 422, 198-207 (2003)). MALDI-TOF(matrix-assisted laser desorption/ionization coupled with time of flight(TOF)) is a method of choice to be used for proteins (Tanaka, K. Theorigin of macromolecule ionization by laser irradiation (Nobel lecture).Angew Chem Int Ed Engl 42, 3860-70 (2003).). Tandem MS (MS/MS) may beused for selective isolation of peptide fragments to read out the(partial) amino acid sequence, and LC/MS (liquid chromatography coupledto MS) may be used for the identification of small peptides. However,the 2DE/MS detection is restricted to pI between 4 and 10 and proteinswithin an MW range of 10-200 kDa. Thus, peptides or small proteins(0.5-10 kDa), such as hormones, adipokines and growth factors, which arerelated to obesity pathogenesis may not be detected by 2DE/MS. Thus,additional initial separation systems such as C/MS using HPLC coupledMALDI-TOF for differential small peptide display is contemplated for use(America, A. H., Cordewener, J. H., van Geffen, M. H., Lommen, A.,Vissers, J. P., Bino, R. J. & Hall, R. D. Alignment and statisticaldifference analysis of complex peptide data sets generated bymultidimensional LC-MS. Proteomics 6, 641-53 (2006). Surface enhancedlaser desorption ionization and time of flight (SELDI-TOF) usingchromatographic chip surfaces based on amino acid sequence, proteinstructure, charge or hydrophobicity is also contemplated for use(Weinberger, S. R., Dalmasso, E. A. & Fung, E. T. Current achievementsusing ProteinChip Array technology. Curr Opin Chem Biol 6, 86-91(2002)), as well as antibody proteomics based on immunoaffinity(Ingvarsson, J., Lindstedt, M., Borrebaeck, C. A. & Wingren, C. One-stepfractionation of complex proteomes enables detection of low abundantanalytes using antibody-based microarrays. J Proteome Res 5, 170-6(2006).).

TABLE 3 PCR primers for differentiation specific genes Gel Product laneGene Primer sequence (sense/antisense) size (bp) Gen ID # 1 Marker 1 kbplus DNA ladder of 200, 300, 200-1,000 400, 500, 650, 850, and 1000 bp 2PPARγ2 5′-GGATGTCGTGTCTGTGGAGA-3′ (SEQ ID NO:1 630 BC006811 Adipogenic5′-TGAGGAGAGTTACTTGGTCG-3′ (SEQ ID NO:2) 3 LPL5′-GAGATTTCTCTGTATGGCACC-3′ (SEQ ID NO:3) 276 BC011353 Adipogenic3′-CTGCAAATGAGACACTTTCTC-3′ (SEQ ID NO:4) 4 αP25′-GTACCTGGAAACTTGTCTCC-3′ (SEQ ID NO:5) 418 BC007538 Adipogenic5′-GTTCAATGCGAACTTCAGTC-3′ (SEQ ID NO:6) 5 PPARδ5′-GGTGAATGGCCTGCCTCCCTACAA-3′ (SEQ ID NO:7) 380 BC007578 Anti-5′-CACAGAATGATGGCC GCAATGAAT-3′ (SEQ ID NO:8) adipogenic 6 ALP5′-TGGAGCTTCAGAAGCTCAACACCA-3′ (SEQ ID NO:9) 452 BC014139 Osteogenic5′-ATCTCGTTGTCTGAGTACCAGTCC-3′ (SEQ ID NO:10) 7 OC5′-CATGAGAGCCCTCACA-3′ (SEQ ID NO:11) 310 NM199173 Osteogenic5′-AGAGCGACACCTAGAC-3′ (SEQ ID NO:12) 8 28S rRNA5′-GTGCAGATCTTGGTGGTAGTAGC-3′ (SEQ ID NO:13) 589 BC000380 Internal5′-AGAGCCAATCCTTATCCCGAAGTT-3′ (SEQ ID NO:14) Control

TABLE 4 Sequence Identifiers for Relevant Proteins Whose Genes may beModulated by Exposure to Oleuropein or Analogues or Derivatives thereof.GenBank Name of Accession Protein Number DNA/Protein SEQ. I.D. NO.PPAR-gamma isoform 2 NP_056953 Protein 15 Leptin DD247154 DNA 16 P2Adipocyte Protein XM_939801 DNA 17 Adipsin M84526 DNA 18 AngiotensinogenProtein AAD14288 Protein 19 Angiotensinogen DNA BC011519 DNA 20Complement Factor H AAH58009 Protein 21 Complement Factor D BC057807 DNA22 Lipoprotein Lipase M15856 DNA 23 BC011353 DNA 24 NM_000237 DNA 29Perilipin NM_002666 DNA 25 Glucose Transporter 4 NP_001033 Protein 26(GLUT4) SREBF1 NM_004176 DNA 27 CCAAT NM_005194 DNA 28 PlasminogenActivator NP_000593 Protein 30 Inhibitor-1 Adiponectin NM_004797 DNA 31Interleukin-6 NM_000600 DNA 32 Interleukin-6 NP_000591 Protein 33TNF-alpha NP_000585 Protein 34 PPAR-alpha NM_005036 DNA 35 PPAR-alphaNP_005027 Protein 36 PPAR-gamma NM_015869 DNA 37 PPAR-gamma NP_056953Protein 38 PPAR-delta NM_006238 DNA 39 PPAR-delta NP_006229 Protein 40

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Modulation of Adipogenesis and Lipodystrophy by OleuropeinOleuropein Modulates Adipocyte Differentiation Methods

Human mesenchymal stem cells (hMSCs) at 10⁴ cells/cm² were cultured inMSC growth medium first to confluence. Adipogenesis was induced two dayspost confluence by culturing the cells in adipogenic medium (AIM)containing 10 μM dexamethasone, 1 μg/ml insulin, and 0.5 mM3-isobutyl-1-methylxanthine, in the absence and presence of oleuropeinat 1, 10 and 100 μM. Fresh culture medium was changed every 3 days. Fulldifferentiation of hMSC to adipocytes was detected by Oil Red 0 stainingat day 12. Gene expression was monitored by RT-PCR, using primers shownin Table 3.

Results

It was determined that oleuropein down-regulates adipocytedifferentiation, fat accumulation and adipogenic gene expression inhuman mesenchymal stem cells (hMSCs). As seen in FIG. 1, hMSCs grown incontrol medium do not stain with Oil Red O (counterstained withhematoxylin), while hMSCs grown in AIM contain lipid droplets that stainwith Oil Red O. Oleuropein shows dose dependent inhibition of adipocytedifferentiation. We are confining these results using flow cytometry.Oleuropein down-regulates the expression of adipogenic genes PPARγ2, LPL(lipoprotein lipase), and αP2 (lipid binding protein). Oleuropeinup-regulates PPARδ expression while the expression of the internalcontrol 28S rRNA remains relatively constant.

Oleuropein De-Differentiates Adipocytes and Allows Transdifferentiationinto Osteoblasts

Methods

For transdifferentiation studies, hMSCs were cultured in adipogenicmedium for 12 days, allowing full differentiation into adipocytes.Oleuropein (80 nM) was added to the culture medium for 48 hours.De-differentiation of adipocytes was detected by the disappearance ofthe accumulated lipid by Oil Red O staining. Oleuropein-treated cellswere collected by trypsinization and cultured in osteogenic mediumcontaining 10 nM dexamethasone, 10 μM β-glycerophosphate, 50 μg/mlL-ascorbate 2-phosphate, and 10 nM 1α, 25-dihydroxyvitamin D3 for 6days.

Results

As seen in FIG. 2, fully differentiated adipocytes from hMSC lost theirOil Red 0 staining after the addition of oleuropein. Furthermore,treatment with osteogenic medium resulted in transdifferentiation intoosteoblasts. In contrast, hMSC-derived adipocytes treated withosteogenic medium, but not with oleuropein, showed little change at day6. 48 hours after the addition of oleuropein, the expression ofadipogenic marker genes PPARγ2, LPL, and αP2 were down-regulated, whilePPARδ was up-regulated. Upon the switch to osteogenic medium, expressionof osteocalcin (OC) and alkaline phosphatase (ALP) are observed.

Summary of Results

It was determined that oleuropein can modulate adipogenicdifferentiation of cultured hMSC, and can de-differentiate hMSC-derivedadipocytes, allowing transdifferentiation. These results are importantbecause they show that oleuropein can modulate the differentiation andcommitment steps in adipogenesis, and offer a possible cellularmechanism for anti-obesity effects.

Example 2 Modulation of Endothelial Dysfunction and Atherosclerosis byOleuropein

Oleuropein Reduces Diet-Induced Atherosclerosis in the Western Diet-FedapoE ko Mouse

Methods

It was previously demonstrated that oleuropein and OLE, followingadministration to mice, could be detected in the blood and urine byLC-MS. To test whether oleuropein reduces diet-induced atherosclerosis,oleuropein was administered to apoE ko mice at various doses from 0.25mg/ml, 2.5 mg/ml, and 25 mg/ml in the drinking water followed by feedingthem with a Western diet containing 42% of calories from fat(Harlan-Teklad). At the 4 month time-point for the 0.25 mg/ml dose(daily dose 1.25 mg), serum levels of oleuropein achieved with this doseare comparable to those in animals fed a diet supplemented with oliveoil.

The mice were sacrificed, blood and tissues collected. Aortas weredissected from the aortic valve to the iliac bifurcation, openedlongitudinally, and pinned to a black wax surface. Atheroscleroticlesions were stained with Oil Red O. The total area of the aorta and theatherosclerotic lesion areas were determined by planimetry using ImagePro software. FIG. 3 shows representative aortas stained with Oil Red 0from untreated and oleuropein treated apoE ko mice.

Results

As seen in FIG. 4, there was a significant reduction in lesion area at 4months in both male and female apoE ko mice that were treated witholeuropein. Female mice had more lesions than male mice in both controland oleuropein-treated groups, consistent with the known gender effectsin the apoE ko model. Error bars indicate SEM; differences betweencontrol and oleuropein-treated mice were significant for both genders(*) at p<0.05.

Summary of these Results

It was demonstrated that oleuropein modulates diet-induced atherogenesisin the Western diet-fed apoE ko mouse model, at doses comparable tothose achievable by diet. These results suggest that the beneficialeffects of a Mediterranean diet may not be due solely to effects ofolive oil on the lipid profile, but may also be mediated in part bynon-lipid components such as oleuropein.

Example 3 Computational Approaches to New Treatments for Obesity Methods

Detailed theoretical and computational analysis for binding ofoleuropein to PPARδ, PPARγ and PPARα was performed, using an approachthat combines AutoDock, MD and MM-PBSA calculations. We used the crystalstructure of PPARδ and PPARγ ligand binding domains. The largeligand-binding site is formed by several α-helices, including theC-terminal AF-2 helix. We first used molecular docking to generateseveral distinct binding orientations, and performed molecular dynamicssimulation to further relax the complex. Then, we applied MM-PBSA toestimate the affinity for each binding mode. The binding modes with thelowest free energy are expected to be the most favorable. We thenanalyzed the detailed interactions based on these binding modes.

Oleuropein Mimics the Binding Mode of High Affinity Ligands for PPARδ

FIG. 5 a shows docking of oleuropein to PPARδ in the energetically mostfavorable binding mode (II), with free energy of −52.73 kcal/mol. In thepredicted structure, oleuropein occupies the Y-shaped ligand-bindingpocket, identical to two known ligands of PPARδ: the synthetic agonistGW2433, which is a high-affinity ligand, and the natural fatty acideicosapentanoic acid (EPA), which binds efficiently to all three PPARs.

FIG. 5 b shows an overlay of oleuropein (red and blue), GW2433 (green)and EPA (purple) in their bound configurations. Oleuropein's sugar groupis located in a similar position and orientation as the carboxyl groupsin GW2433 and EPA. The two hydroxyls on the sugar ring are orientedtoward the AF-2 helix and held in place through a network of hydrogenbonds with Y473, and hydrophobic interactions with L469 and T84, as seenin FIG. 5 d. Both Y473 and L469 are part of the AF-2 helix. The samenetwork of hydrogen bonds occurs in the binding of ligands to PPARδ andy, and is believed to be important for ligand-mediated activation ofthese receptors.

Oleuropein Shows Different Binding Modes than High Affinity Ligands forPPARγ

FIG. 6 shows docking of oleuropein with PPARγ in the most favorablebinding mode (I), with free energy of −27.09 kcal/mol. In the predictedstructure, oleuropein binds into the large PPARγ pocket in an extended(up-down) conformation rather than the U-shaped conformation of otherknown ligands such as G1262570 and rosiglitazone. FIG. 6 b shows anoverlay of oleuropein (red and blue), rosiglitazone (yellow) and thePPARγ agonist G1262570 (green). Unlike known ligands, oleuropein doesnot interact directly with AF-2 helix. At one end of the molecule, thedihydroxyl phenol group is directed into the solvent-accessible channelbetween H3 and the β strands. At the other end, the oleuropein sugarheadgroup is inserted 4.0 Å deeper than the phenyloxazole tail ofGI262570 into the lipophilic pocket adjacent to the β sheet, forming twostrong hydrogen bonds with the carboxyl group of E259 (shown in FIG. 6d).

Oleuropein Shows Different Binding Modes than High Affinity Ligands forPPARα

FIG. 7 a shows the binding structure of PPARα-oleuropein complex withfree energy of −24.71 kcal/mole. The PPARα backbone is represented bythe yellow ribbon, and oleuropein is represented with vdw and is colorcoded as follows: carbon, cyan and oxygen, red. In the predictedstructure as shown, oleuropein adopts a Y-shaped configuration when itbinds to PPARα, similar to how it binds to PPARδ. This is consistentwith the similarities between the binding sites of PPARα and PPARδ. Thisconformation differs from the U-shaped conformation of other knownligands of PPARα, such as GW409544. FIG. 7 b represents superposition ofthe structures of GW409544 (green) and oleuropein (red and blue) boundto PPARα. FIG. 7 c shows the chemical structures of oleuropein andGW409544. FIG. 7 d shows hydrogen bonds formed by oleuropein and thesurroundings (indicated as green dotted lines).

Summary of these Results

The known ligand binding domains of PPAR δ is large and Y-shaped. Ourmolecular modeling results show binding modes for oleuropein that are asfavorable as the currently known ligands. Oleuropein mimics preciselythe binding mode of high affinity ligands for PPARδ, indicates thatoleuropein is capable of modulating PPARδ activity similar to that ofknow PPARδ agonist. Oleruopein has the potential to increase fatoxidation, increase energy uncoupling and thermogenesis and thusdecrease fat accumulation, reduce body weight and prevent obesity. ForPPARγ, on the other hand, in the predicted structure, oleuropein bindsto PPARγ in an extended (up-down) conformation rather than the U-shapedconformation of other known ligands such as G1262570 and rosiglitazone.Unlike these ligands, oleuropein does not interact directly with AF-2helix. These results predicate that oleuropein has the potential tointeract with PPARγ distinct from PPARγ known agonists including TDZ.Oleruopein does not act on the AF2 helix of PPARγ, the region thatinvolves in adipogenesis, thus suggesting an important mechanism forregulation of PPARγ activity in uncoupling adipogenesis from insulinsensitivity and other activities. For PPARα, in the predicted structure,oleuropein adopts a Y-shaped configuration when it binds to PPARα. Thisis similar to how it binds to PPARδ. This is consistent with thesimilarities between the binding sites of PPARα and PPARδ as well astheir functions. Both PPARα and PPARδ are involved in fat oxidation andenergy utilization. In this context, the energetically favorable Yconfiguration of oleuropein may offer insight into its biologicaleffects. Studies are underway to confirm the biological significance ofthese modeling results, and correlating them with in vitro and in vivodata. We are studying the experimental binding and co-crystallization ofoleuropein with the ligand binding domains of PPAR α, δ and γ. We areanalyzing the expression and activity of PPAR α, δ and γ in tissues fromoleuropein-treated apoE ko mice that show reduction in diet-inducedatherosclerosis, to identify the importance of these isoforms to theobserved biological activity.

Example 4 Preparation of Oleuropein from Olive Leaf Extract LC-MSStandardization of Olive Leaf Extract (OLE) Containing Oleuropein as theActive Moiety A. Preparation of Olive Leaf Extract

Selection cleaning and processessing of the olive leaves. Healthy wholeolive leaves were selected and cleaned to remove dust, residualinsecticides and contaminating material. The leaves were cut into smallpieces or powdered into fine powders and placed in a sterile flask justbefore extraction, with sterile distilled water and pre-heated to 80° C.

Extraction media and ratio. A comparison was made between the extractionwith water, PBS (Phosphate Buffered Saline, 10 mM sodium phosphatebuffer, pH 6.8, containing 0.15% NaCl) and organic solvents (50%methanol, or 40% ethanol). No significant differences were found inbiological activity using the different extraction methods. Since thetherapeutic ingredients in olive leaf are apparently water-soluble, adecision was made to use water extraction, because it gives stableextracts that can be concentrated with ease. For efficient extraction,it was determined that the optimal ratio is one gram of dry leaves to 40ml of water.

Extraction conditions: The extraction mixture was covered and incubatedwith agitation for 10-12 hours at 80° C. in a water bath. It isimportant that the extraction temperature be kept under 85° C. Heatingabove 86° C. inactivates oleuropein, one of the principle ingredients ofOLE. Alternative methods for extraction including but not limited tomicrowave for two repeats of 5-10 min, or ultrasound for 25 min.

Concentration of the extract by lyophilization. At the end of 10-12hours at 80° C., proper extraction results in a medium brown colored OLEwith about 70-80% of the original volume. The liquid was poured off andcollected. The leaves were extracted again for a second time under thesame conditions to ensure complete extraction of the active ingredients.Small pieces of leaves and insoluble material in the extract wereremoved by centrifugation at 20,000×g for 30 min. The clear supernatantwas collected and labeled as Step 1 OLE. Step 1 OLE was concentrated byfreeze-drying using a Lyophilizer. The dried OLE was collected, andlabeled as Step 2 OLE.

Sterilization by Millipore filtration. Step 2 OLE was dissolved insterile water at 10-20 mg/ml, sterilized by Millipore filtration with a0.45 micron filter, distributed at small lots in sterile cryo-tubesunder aseptic conditions and stored at −80° C. This is Step 3 OLE andits oleuropein content was quantitated by LC-MS using known oleuropeinstandard. Standardized OLE was used in all of our experiments.

Preparation and LC-MS Standardization of Oleuropein

Oleuropein was prepared and quantitated by LC-MS as noted above. Thisprocess was performed using an HP1100 equipped with diode array detectorand ESI-mass spectrometer. LC was done using a C18 column, using agradient of 5-95% CH₃CN—H₂O containing 1% acetic acid. The diode arrayrecordings were made at 280 nm and 230 nm, and the ESI mass spectrum wasmade in negative detection mode. Purified oleuropein, was used as astandard. Experiments were optimized by infusion of the standards innegative scan mode to investigate the [M-H] ion of oleuropein glycoside(m/z 539). Oleuropein fractionates as a single peak by HPLC at 1.846min, and contains predominantly oleuropein glycoside (m/z 539). Theoleuropein content in the lyophilized OLE ranges from 20-25% asstandardized by LC-MS.

Preparation and LC-MS Standardization of Hydroxytyrosol

Hydroxytyrosol was prepared from homogeneous oleuropein isolated fromOLE. The procedure involves treatment of oleuropein with β-glycosidase(including but not limited to Sigma G4511) in 80 mM sodium acetate pH5.0 using 1 Unit of enzyme/μmole of substrate at 37° C. for 1 hr toremove the glucose moiety and to yield oleuropein aglycone. Theoleuropein aglycone was then be treated with esterase (including but notlimited to Sigma E0887) in sodium phosphate buffer at pH 7.5 using 1Unit of enzyme/μmole of substrate at 25° C. for 1 hr to yieldhydroxytyrosol and elonelic acid. The final products were resolved andquantitated by LC-MS as described above. Detection and quantificationwere performed at 280 and 320 nm for hydroxytyrosol at 0.698 min (m/z153) and oleuropein aglycone at 2.087 min (m/z 377), while 240 nm wasused for the detection of elenolic acid at 1.846 min (m/z 241).

LC-MS Standards

Synthesis of Hydroxytyrosol

Hydroxytyrosol, the major metabolite of oleuropein is biological active.We have designed a unique method for chemical synthesis ofhydroxytyrosol. Chemically synthesized hydroxytyrosol is as active asits natural counterpart from OLE. Major steps in chemical synthesis ofhydroxytyrosol are briefly outlined below:

We started our hydroxytyrosol synthesis from 3,4-dihydroxylphenylaceticester. It was prepared by the following procedure. The reaction mixtureof 3,4-dihydroxylphenylacetic acid 168 mg in CH₃OH was treated withAcetyl chloride and CH₃OH mixture. The reaction mixture was stirred atroom temperature overnight. The reaction mixture was purified by columnchromatography. A total of 140 mg of oily compound was obtained

Hydroxytyrosol was prepared from 3,4-dihydroxylphenylacetic ester byLiAlH₄ reduction. 140 mg of 3,4-dihydroxylphenylacetic ester wasdissolved in tetrahydrofuran (THF). 1M of LiAlH₄ (4 ml) solution wasadded to the reaction mixture. The mixture was stirred at 0° C. for 2hours. The reaction mixture was purified by column chromatography togive hydroxyltyrosol and characterized by LC-MS.

B. HPLC Analysis of OLE Components

Step 3 OLE was subjected to HPLC using a Waters two solvents deliverysystem with photodiode array detector. A Symmetry C18 column (5 μm,3.9×250 mm) column with a Sentry Guard 3.9×20 mm insert was used. Dataacquisition and quantitation were performed with Millennium 32 software(version 3.0). The mobile phase was 79% distilled water and 21%acetonitrile (HPLC grade), both acidified to pH 3 with 0.1 Morthophosphoric acid. This solvent system is designed for resolution andquantitation of polypheolic compounds. The flow rate was 1 ml/min, andthe injection volume was 20 μl. Polyphenolic compounds were monitored byabsorbance at 280 in. The run time was 35 minutes. The structure ofoleuropein is shown in FIG. 8. The elution profile is shown in FIG. 9. Atotal of seven major peaks were resolved from the included material. Thesolvent front and excluded material appear before peak 1, with retentiontime (Rt) less than 6 minutes.

C. Identification of OLE Components

The major OLE components were identified by TLC and HPLC with knownstandards. TLC was carried out on silica gel 60 F254 (Merck) withchloroform/methanol/acetic acid (70:30:10). Secoiridoids and flavonoidswere detected by visualization under UV light at 254 nm with 10% ferricchloride and 10% aminoethyl diphenylborate spray, using known standards.HPLC verification was conducted by repeated HPLC of peak material withknown amounts of standards. The identities of the polyphenolic compoundsin each peak are shown in Table 5, along with their relative levels inthe OLE and their cytotoxicity.

TABLE 5 Identity and characterizations of phenolic compounds in OLE PeakRt (min) Compound % (w/w) Cytotoxicity Excl. <6 Solvent front — — 1 6.6Rutin 0.34 — 2 7.8 Verbascoside 0.38 — 3 8.9 Luteolin7-glucoside 0.68 —4 16.0 Apigenin7-glucoside 0.18 — 5 17.0 Flavonid x 0.56 — 6 22.8Oleuropein 12.8 — 7 34.0 Oleuroside 0.51 —

D. LC-MS Analysis

Samples: The standard and peak 6 oleuropein were prepared in sterilewater with a final concentration of 1 mg/ml. Step 3 OLE was prepared asdescribed above at a final concentration of 10 mg/ml. All samples wereMillipore filtered and stored at −20° C. until use.

Experimental protocol: LC-MS was performed using an HP1100 equipped withdiodearray detector and ESI-mass spectrometer. LC was done using a C18column (20×4.0 mm), with 4 minute elution using a gradient of 5-95%CH₃CN (containing 1% acetic acid)-H2O (containing 1% acetic acid). Thediodearray records were made at 280 nm and 230 nm, and ESI mass spectrumwas made in negative detection mode. The standard and peak 6 oleuropeinwere diluted to a final concentration of 0.5 mg/ml, and OLE was dilutedto 5 mg/ml. The injection volumes were all 5 μl. Experiments wereoptimized by infusion of the standards in negative scan mode toinvestigate the [M-H] ion of oleuropein (m/z 539).

Results

Peak 6 Oleuropein Showed Similar Purity to the Oleuropein Standard by LC

FIG. 11 shows the LC-MS results of oleuropein standard (11A), peak 6oleuropein (11B) and OLE (11C). As seen in FIGS. 11A-LC and 11B-LC, peak6 oleuropein is as pure as the standard. A single major peak wasobserved at 1.827 min and a minor peak at 1.696 min (less than 1%) inboth samples.

Peak 6 Oleuropein Showed a Single Mass as the Oleuropein Standard by MS

MS of the standard and the peak 6 oleuropein are seen in FIGS. 11A-MSand 11B-MS. A single mass peak at [M-H]⁻=539 corresponds to oleuropeinwas detected in both samples. A minor peaks was observed at mass[M-H]⁻=1079 it represents [2M-H].

OLE Contains Several Components by LC

FIG. 11C-LC represents the LC profile of OLE. Several peaks weredetected. Peaks A at 1.82 min is the major peak, peaks B, and C aremoderate and minor respectively.

MS of OLE Identified Oleuropein, Oleuropein Aglycone and Hydroxytyrosol.

FIGS. 11C-MS of OLE A, OLE B, and OLE C represent MS of LC peaks A, B, Crespectively. MS of peaks A, B, and C identified mass peaks at[M-H]⁻=539, 377 and 153 correspond to oleuropein, oleuropein aglyconeand hydroxytyrosol respectively.

Heavy metals, pesticides, fungicides & herbicides were analyzed and notdetected in the samples.

Summary and Discussion of Results:

LC-MS results show that peak 6 oleuropein consists of a single majorpeak at [M-H]⁻=539 corresponds to oleuropein. The purity of this sampleis equal to or even better than the standard. The relative size of thepeak [M-H]⁻=1079 is less in peak 6 oleuropein than in the standard.

LC-MS of OLE indicates that in addition to oleuropein, oleuropeinaglycone and hydroxytyrosol are also present. Oleuropein is aheterosidic ester of elenolic acid and hydroxytyrosol(3,4-dihydroxy-phenylethanol), containing a molecule of glucose, uponβ-glycosidase action it yields oleuropein agylcone. Hydrolysis ofoleuropein aglycon yields hydroxytyrosol and elenolic acid. Thestructural relationships among these compounds are shown in FIG. 10.These molecules are major metabolites of oleuropein thus their presencein the OLE is expected. Elenolic acid has no UV absorbance thus itcannot be detected in LC-MS analysis.

Oleuropein can also be prepared from olive leaves by extracting with 50%aqueous methanol. After evaporation of methanol, the aqueous phase canbe extracted with chloroform and then saturated with NaCl and filtered.Oleuropein and other phenolic compounds can be extracted with ethylacetate. The ethyl acetate phase was totally evaporated to dryness.Oleuropein can be further purified and characterized by LC-MS asdescribed above.

Example 5 Oleuropein is Bioavailable: it is Absorbed and Well Toleratedin Animals A. Bioavailability Studies

It is important to establish that chronic administration of oleuropeinresults in the absorption of the biologically active material in vivo.In addition, it is necessary to determine the physiologically achievableand relevant concentrations in tissue, blood, and urine, and to comparethese with concentrations that show activity in vitro or in cellculture.

To verify that we can administer oleuropein chronically to mice, weadded purified oleuropein to drinking water at concentrations of 5μg/ml, 50 μg/ml, 500 μg/ml, and 5 mg/ml. The water intake of miceaverages 5 ml per mouse per day, these dosages of oleuropein result indaily doses of 0.025 mg, 0.25 mg, 2.5 mg, and 25 mg per mouse per day(equivalent to 1, 10, 100, and 1000 mg/kg body weight). 5 male and 5female C57BL/6 wild-type mice were used for each dose of oleuropein, andhoused them in metabolic cages that allow precise determination of waterintake and quantitative urine collection.

B. LC-MS Quantitation

After seven days to 4 months of chronic steady-state administration,mice blood and urine were collected. 100 μl of serum or urine wereextracted with 1 ml ethyl acetate, evaporated to dryness, andreconstituted with 25 μl water and 5 μl was injected for LC-MS analysis.

LC-MS quantitation was performed using an HP1100 equipped with diodearray detector and ESI-mass spectrometer. LC was done using a C18 column(4×20 mm), with a 4 minute elution using a gradient of 5-95% CH₃CN—H₂Ocontaining 1% acetic acid. The diode array recordings were made at 280nm and 230 nm, and the ESI mass spectrum was made in negative detectionmode. Purified oleuropein, and hydroxytyrosol (synthesized by us) wereused as standards. The injection volumes were 5 μl. Experiments wereoptimized by infusion of the standards in negative scan mode toinvestigate the [M-H] ion of oleuropein glycoside (m/z 539), oleuropeinaglycone (m/z 377), and hydroxytyrosol (m/z 153).

C. Summary of Results

FIG. 12 shows results from LC-MS analysis of mice blood and urine after4 months of chronic steady-state administration. Oleuropein standardshows a single peak by LC (12A) with mass of 539 (m/z 539) (12B). Incomparison, the LC profile of serum (12C-D) and urine (12E-F) containmainly hydroxytyrosol (m/z 153) as well as other derivatives andmetabolites. Our results show that 1) we can administer oleuropein in adose-dependent manner chronically to mice, 2) oleuropein is absorbed bythe mice and metabolized to hydroxytyrosol, and 3) we can quantitate theamount of oleuropein and hydroxytyrosol in the serum and urine of miceby LC-MS.

In summary, our results show that oleuropein is bioavailable. It isabsorbed and well tolerated in animals. It is metabolized tohydroxytyrosol, which is secreted in urine.

Example 6 Inhibition of HIV-1 Fusion by Oleuropein and HydroxytyrosolMaterials and Methods Oleuropein (Ole) and Hydroxytyrosol (HT)

Oleuropein (Ole) was purified from olive leaf extract, characterized,and standardized by liquid chromatography-coupled mass spectrometry(LC-MS) [3]. Hydroxytyrosol (HT) was prepared by stepwise hydrolysis ofOle with β-glucosidase (Sigma G4511) in 80 μM sodium acetate, pH 5.0using 1 Unit/μmole substrate at 37° C. for 1 hr. This treatment removesthe glucose moiety from Ole and yields oleuropein aglycone (Ole-AG).Ole-AG was subsequently hydrolyzed with esterase (Sigma E0887) in 50 mMsodium phosphate buffer at pH 7.5 at 1 Unit/μmole substrate at 25° C.for 1 hr to yield hydroxytyrosol and elenolic acid. The mixture wasseparated by HPLC and standardized by LC-MS. HT was also prepared bychemical synthesis from 3,4-dihydroxylphenylacetic ester (DHPA).

Cell Lines and HIV-1

Uninfected MT2 and H9 cell lines, and HIV-1_(IIIB) chronically infectedH9 (H9/HIV-1_(IIIB)) and HIV-1/IIIB virus, were obtained through theAIDS Research and Reference Reagent Program, NIAID, NIH. MT-2 cells[4,5] were obtained from D. Richman, and H9 and HIV-1_(IIIB) virusstocks [6,7] from R. Gallo. The cell lines were cultured in RPMI medium1640 containing 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mML-glutamine, and 10% heat-inactivated fetal calf serum. Viral stockswere prepared and standardized as described [8].

Anti-HIV and Cytotoxicity Assays

The effects of Ole and HT on acute HIV infection and viral replicationwere measured by assays on syncytial formation in cell-cell HIV-1transmission and on HIV-1 core protein p24 expression as described [8].Cytotoxicity was evaluated by the MTT assay [8].

Molecular Modeling

Molecular modeling was performed by molecular docking, moleculardynamics (MD) simulation and free energy calculations [9,10]. Dockingwas performed with Autodock version 3.0.5 [11]. The relaxation ofdocking structure obtained was then implemented under Discovery fromInsight II (Accelrys Inc., San Diego, Calif., U.S.A.) using 500 steps ofSteepest Descent followed by Conjugate Gradient until the root meansquare of the energy gradient reaches a value of 0.01 kcal/molÅ.

HIV-1 gp41 Fusion Peptides C34 and N36

HIV-1 gp41 fusion peptides, N36 and C34 were synthesized by solid phaseFMOC method (GeneMed, CA) and purified by HPLC. The sequences of thesepeptides are (Ac-SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL-NH₂) and(Ac-WMEWDRFINNYTSLIHSLIEESQNQQEKNEQELL-NH₂) respectively. Correspondingviral peptides N36 and C34 prepared from HIV-1 were obtained through theNIH AIDS Research and Reference Reagent Program, NIAID, NIH [12] andused as standards for purification and bioassays.

Fusion Complex Formation

Fusion complex formation was carried out by incubating equimolar amountsof HIV-1 gp41 fusion peptides N36 and C34 in PBS (Phosphate BufferSaline, containing 50 mM sodium phosphate, pH 7.2 and 150 mM NaCl) at 10or 20 μM each at 37° C. Peptide N36 was first incubated either alone orwith various concentrations of Ole or HT for 30 min. Next, an equimolaramount of C34 was added and the samples incubated for 30 min.

Native Polyacrylamide Gel Electrophoresis (N-PAGE)

N-PAGE was carried out as previously reported [13] with modificationsthat involve fusion peptide concentration, order of reactions, and timeof incubation in fusion complex formation. Tris-glycine gels (18%,Invitrogen, Carlsbad, Calif.) were electrophoresed at 120 V for 2 h,stained with Coomassie Blue and analyzed by densitometry.

Circular Dichroism (CD) Spectroscopy

CD spectra were recorded on an AVIV 62-DS CD spectrometer, using 1 mmsample cells and a fixed temperature of 4° C. [8]. Each spectrum is asmoothed average of 10 scans. The bandwidth for each measurement was 1nm. CD intensities are expressed as mean residue ellipticities [0](degrees cm²/dmol). Prior to calculation of the final ellipticity, allspectra were corrected by subtracting the reference spectra of PBSwithout peptides.

Results Preparation and LC-MS Analysis of Oleuropein (Ole) andHydroxytyrosol (HT)

FIGS. 13A-1B and 13C-1D represent LC-MS analysis of Ole and HT. Olefractionates as a single peak by HPLC at 1.848 min with m/z of 539 andHT as a single peak by HPLC at 1.108 min with m/z of 153. FIG. 13E showsthe major steps in Ole metabolism. These are also the basic reactions inHT preparation from Ole. First, the glucose moiety is removed byβ-glycosidase to yield oleuropein aglycone (Ole-AG). Next, Ole-AG ishydrolyzed by esterase to yield HT and elenolic acid (EA). FIG. 13Fshows chemical synthesis of HT using 3,4-dihydroxylphenylacetic acid(DHPAA) as the starting material. Two major steps are involved: 1)acetylation with acetyl chloride (AcCl), and 2) reduction by LiAlH₄ toyield HT. Chemically synthesized HT was purified and characterized byLC-MS, and demonstrates identical biological, chemical and physicalproperties as natural HT prepared from Ole from olive leaf extract.

OLE and HT Inhibit HIV-1 Infection and Replication, but are not Toxic toTarget Cells

Ole and HT exhibit dose dependent inhibition of HIV-1 infection andreplication as measured by syncytial formation and p24 production (Table6). The average EC₅₀ for Ole is 55 μM for syncytial formation and 73 nMfor p24 production. The corresponding EC₅₀s are 61 nM and 68 nM for HT.No cytotoxicity was detected, either by MTT assay or trypan blue dyeexclusion, over a 10,000-fold concentration range from 1 nM to 10 μM.The EC₅₀s for inhibition on fusion complex, 6HB formation are alsopresented in Table 6.

Ole and HT Bind to the Conserved Hydrophobic Pocket on the Surface ofthe Central Trimeric Coiled-Coil of HIV-1 gp41

Inhibition of syncytial formation by Ole and HT reflects effects onearly events during viral infection/entry, including CD4 receptor andcoreceptor binding as well as viral fusion. To probe anti-HIV mechanismsof Ole and HT, we carried out molecular docking and MD calculations ofthese small molecules with viral targets. We found that Ole and HT bindto the conserved hydrophobic pocket on the surface of the centraltrimeric coiled-coil of the HIV-1 gp41 fusion domain.

HIV-1 envelope glycoprotein (Env) mediates viral entry by fusing virusto target cells. Env is trimeric on the virion surface. Each monomercontains a surface subunit, gp120, for virus binding to CD4 receptor andcoreceptors [14-17] and a noncovalently associated transmembranesubunit, gp41, that mediates fusion of the virus with the target cell[18,19]. FIG. 14A shows the structure of HIV-1 gp41. Like other type Itransmembrane proteins, HIV-1 gp41 consists of extracellular(ectodomain), transmembrane, and cytoplasmic domains. The ectodomaincontains four functional regions: the fusion peptide, N-terminal heptadrepeat (NHR), C-terminal heptad repeat (CHR), and a tryptophan-richregion. Binding of gp120 to the cellular receptor CD4 and co-receptortriggers conformational changes in gp41 that induce fusion [20-23]. Thisincreases exposure of two heptad repeat motifs, NHR and CHR, andinsertion of the fusion peptide into the target membrane [20-23].Subsequently NHR and CHR fold in an antiparallel manner to create thesix-helix bundle 6HB composed of a trimeric NHR coiled-coil coresurrounded by three CHR helices that pack in the grooves of thecoiled-coil as seen in FIG. 14B [24-26]. Formation of the 6HB promotesfusion between viral and cellular membranes and is essential for viralentry and infection [25,26]. Ole and HT interact with the NHRcoiled-coil trimer N36 helices and interfere with the formation of 6HBwith the CHR, C34, as shown in FIGS. 14C and 14D.

We used the crystal structure of the HIV-1 gp41 fusion complex, PDB code1 AIK [20] as a reference for our modeling work. To provide a ligandbinding site, one of the C34 helices was removed from the 6HB (FIG.15A). FIG. 15B shows the chemical structure of Ole with the 9 freerotatable bonds selected in our modeling interaction.

Molecular simulations suggest that the conserved hydrophobic cavity ofthe gp41 N36 trimer coiled-coil is the most likely binding site for Oleand HT. This cavity is mainly occupied by W628, W631 and neighboring1635 and D632. The predicted binding structures of Ole and HT are shownin FIGS. 15C and 15D respectively. Ole and HT form stable hydrogen bondswith Q577 on the N36 peptide. FIGS. 15E and 15F are ribbonrepresentations of the predicted binding site of Ole and HT. 5HB,consisting of three N36-peptides (pink, residues 546-581) and two C34peptides (green, residues 628-661), is used for docking calculations.Only one groove is exposed for the binding of small molecules. Both Oleand HT occupy the binding site similarly, with the diphenol ring formingstable hydrogen bonds with Q577. This blocks the close contacts betweenthe hydrophobic groove in the gp41 NHR and the indole rings of W631 andW628, thus interfering with the formation of 6HB. In addition tohydrogen binding, hydrophobic interactions with I573, G 572, and L 568also play important roles in the interaction.

Native PAGE Shows that Ole and HT Inhibit HIV-1 Fusion Core 6HBFormation

To test the predictions from molecular modeling, we examined the effectof Ole and HT on the formation of fusion complex 6HB by electrophoreticmobility shift using native-PAGE (FIG. 16A). The electrodes wereconnected from cathode (negative terminal on top) to anode (positiveterminal on bottom) and the peptides move in the electric fieldaccording to their charge and size. Peptides carrying net negativecharges, such as C34, moves toward the positive terminal (bottom)whereas peptides carrying net positive charges, such as N36, movestoward the negative terminal or remain at the top of the well.Incubation of N36 and C34 resulted in the formation of the 6HB fusioncomplex which is larger than C34, moves slower than free C34, and thusmigrates to the middle of the gel. Pre-incubation of N36 with Ole or HTresults in inhibition of 6HB formation. A dose-dependent disappearanceof 6HB band was detected with concomitant appearance of the free C34band. Total inhibition is achieved at 100 nM Ole or HT with EC₅₀s around66 and 58 nM. These results confirm the predictions from molecularmodeling.

CD Analysis Indicates that Ole and HT Inhibit HIV-1 Fusion Core 6HBFormation

We also used CD analysis to confirm molecular modeling predictions (FIG.16B). Because N36 and C34 are single stranded random coils, they do notassume ordered structure in solution, so they display characteristicrandom coil CD spectra. However, formation of fusion complex 6HB resultsin a distinctive CD spectrum, including a saddle-shaped negative peakbetween 210-220 nm in the far UV region and a significant increase inmolar ellipticity (θ) at 222 nm. Preincubation of N36 with Ole or HTinterrupts 6HB formation and results in a dose dependent shift of the CDspectra from helical to random coil with EC₅₀s of 62 nM for Ole and 60nM for HT.

TABLE 6 Anti-HIV Activity and Inhibition on HIV-1 Fusion Core FormationInhibitory Anti-HIV Activity^(a) Activity^(b) EC₅₀ (nM) EC₅₀ (nM) IC₅₀(nM) Fusion Core Formation Syn- Cyto- 6HB (N36 + C34) Compound cytiumP24 toxicity N-PAGE CDSA Ole 55 ± 5 73 ± 8 >10,000 66 ± 5 62 ± 6 HT 61 ±6 68 ± 8 >10,000 58 ± 8 60 ± 4 ^(a)EC₅₀, effective concentration at 50%inhibition; IC₅₀, cytotoxicity concentration at 50% inhibition.^(b)N-PAGE, native polyacrylamide gel electrophoresis; CDSA, circulardichroism spectroscopy analysis. Values are means ± SD of triplicates inthree independent determinations.

Discussion

Ole and HT are small molecules with molecular weights of 539 and 153respectively. Their inhibition of the fusion-promoting refolding of gp41is an excellent example of how small molecules can block formation ofprotein-protein complexes. We narrowed down the target of binding to ahydrophobic pocket on the gp41 inner core. This pocket is highlyconserved among the different HIV clades. Consistent with this, we foundthat Ole and HT are active against a panel of HIV-1 primary isolatesthat includes both M and T tropic strains from different clades. Ourresults suggest that Ole and HT may be useful against other viruses withtype I transmembrane envelope glycoprotein, including severe acuterespiratory syndrome associated coronavirus [23,27], respiratorysyncytial virus, Ebola virus [28], measles virus [29], and avian flu[30,31].

Fuzeon (T-20 or Enfuvirtide) is the only FDA approved HIV fusioninhibitor [32,33]. It is a peptide derived from the CHR region of gp41that partially overlaps with the C34 sequence. Fuzeon is commerciallyproduced by chemical synthesis. Because of its large size—it consists of36 amino acids with a molecular weight of 4492—its manufacturing processis very complex, involving 106 chemical steps [34,35]. In contrast, ourtypical process for chemical synthesis of HT involves only two steps:acetylation and reduction (FIG. 13F). In addition, Ole and HT can alsobe easily prepared from natural olive leaf extract in only two steps:deglycosylation and oxidation (FIG. 13E). The fact that Ole and HT actboth outside and inside of the cellular environments in viral entry andintegration offers unique benefits to these small molecules againstviral resistance.

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Example 7 Effect of Oleuropein and Hydroxytyrosol on HIV-1 IntegraseMaterial and Methods Oleuropein (Ole) and Hydroxytyrosol (HT)

Ole and HT used in this study were prepared and purified from olive leafextract as described in the preceding article.

Target Cells, HIV-1, Anti-HIV and Cytotoxicity Assays

Target cells MT2, H9, HIV-1_(IIIB) chronically infected H9(H9/HIV-1_(IIIB)) and HIV-1/IIIB virus, were obtained through the AIDSResearch and Reference Reagent Program, NIAID, NIH. MT-2 cells were fromD. Richman [7,8]. H9 cells and HIV-1IIIB virus stocks were from R. Gallo[9,10]. The cell lines were cultured in RPMI medium 1640 containing 100U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and 10%heat-inactivated fetal calf serum. Viral stocks were prepared andstandardized as described previously [11]. Anti-HIV activity wasmeasured by the microtiter syncytial formation and HIV-1 p24 productionassays [11]. Cytotoxicity was determined by the MTT assay [11].

Molecular Modeling

A combination of molecular docking, molecular dynamics (MD) simulationand free energy calculations [12,13] were performed to probe theinteractions of Ole or HT with viral targets. Docking was performed withAutoDock version 3.0.5 [14]. Relaxation of docking structure obtainedwas carried out by the program Discovery from Insight II (Accelrys Inc.,San Diego, Calif., U.S.A.), using 500 steps of Steepest Descent followedby Conjugate Gradient until the root mean square (RMS) of the energygradient reaches 0.01 kcal/molÅ.

HIV-1 Integrase

HIV-1 integrase was expressed in E. coli from pIN (F185H/C280S) andpurified according to previously reported method [15]. This recombinantclone makes the integrase protein more soluble and stable withoutaffecting in vitro activity. HIV-1 integrase protein (F185H/C280S) wasused as a standard for purification and assay. The integrase clone pIN(F185H/C280S) and the standard integrase protein were obtained throughthe NIH AIDS Research and Reference Reagent Program, NIAID, NIH from Dr.Robert Craigie [16].

Integrase Substrates

Oligonucleotide substrates were synthesized and purified as describedpreviously [17]. Three types of substrates were synthesized withsequences that correspond to the U3 and U5 ends of HIV-LTR: (i) the21-nucleotide minus strand of U3 end HIV-LTR 5′-GAGTGAATTAGCCCTTCCAGT-3′(SEQ ID NO: 41), and the U5 HIV-LTR, 5′-GTGTGGAAAATCTCTAGCAGT-3′ (SEQ IDNO: 42), as well as their complementary strands, for assaying the3′-processing reaction; (ii) the 19-mer U3-GT, and U5-GT (i.e., U3 andU5 minus the 3′-end dinucleotide GT) for assaying heterologousintegration (strand transfer); and (iii) a 38-mer dumbbell substratewith the sequence 5′-TGCTAGTTCTAGCAGGCCCTTGGGCCGGCGCTTGCGCC-3′ (SEQ IDNO: 43), for assaying the dis-integration.

Radiolabeling and Preparation of the Substrates

The integrase substrates were 5′-end radiolabeled as reported previously[17]. Briefly, 1 g of the oligonucleotide was 5′-end labeled with 100μCi of [α-³²P]ATP (3000 Ci/mmol; 1 Ci=37 GBq; A-mersham) by 20 units ofpolynucleotide T4 kinase in a final volume of 40 μl of kinase buffer(Boehringer Mannheim) at 37° C. for 60 min. The reaction was stopped byEDTA (25 mM) and heat inactivation. Unincorporated label was removed bytwo passages through a Sephadex G-25 spin column (Boehringer Mannheim).The purified labeled oligonucleotide was then annealed with an equimolaramount of unlabeled complementary strand in 10 mM Tris HCl, pH 7.5/1 mMEDTA/100 mM NaCl at 95° C. for 5 min followed by slow cooling. Thedumbbell substrate was self-annealed under the same conditions.

Integrase Assays

Integrase assays were carried out in 20 mM Hepes, pH 7.5/10 mM MgCl₂ orMnCl₂/10 mM dithiothreitol/0.05% Nonidet P-40 (integrase buffer) with 40μmol of HIV-1 integrase and 20 ng of 5′-end radiolabeled substratesspecific for 3′-processing (21-mer U3), strand-transfer (19-mer U3-GT))or disintegration (38-mer dumbbell) in the presence or absence of Ole orHT in a final volume of 10 μl at 37° C. for 60 min. For 3′-processingand disintegration, the reactions were stopped by the addition of 10 μlof 90% formamide/0.025% bromophenol blue/0.025% xylene cyanol/89 nMTris/89 mM boric acid/2 mM EDTA, pH 8.0. Samples were heated at 75° C.for 3 min, load at 10 μl/well onto 18% polyacrylamide denaturing (7.5 Murea) gels in TBE buffer and electrophoresed at 200 V constant voltageat room temperature for 2 h. The results were visualized byautoradiography of wet gels. For strand-transfer (integration), pUC18plasmid DNA (50 ng) was used as the target for the integration of viralDNA into heterologous plasmid DNA. The reaction was stopped by 0.1% SDSand integration products monitored on 1% agarose gels at 10 ul/well inTBE buffer at 100 V constant voltage at room temperature for 45 min. Theresults were visualized by autoradiography of dried gels.

Results Ole and HT Bind to the Catalytic Site of HIV-1 Integrase

To examine the molecular interactions of Ole and HT with HIV integrase,we performed a series of docking simulations. In these studies, wefocused on the catalytic core domain (CCD) of the viral enzyme, becausethe linkages between the CCD and both the N- and C-terminal domains (NTDand CTD) are flexible and have been not precisely determined. We usedthe crystal structure 1QS4 (pdb ID) as the starting structure [18],because it is the only structure has an inhibitor (5CITEP) bound in theactive site. To keep the binding pocket available, we removed the ligand(5CITEP) from the CCD. Before docking, the missing residues in the loopregion and the point mutation at position 185 were restored with AMBERsoftware. The K185 mutation was converted to the native F185.

FIGS. 17A and 17B represent the predicted bound conformations of Ole andHT within the active site of HIV-1 integrase. Two unique binding regionshave been identified within the integrase active site [2, 4], referredto as regions I and II (FIG. 17B). Region I, near the active sitecenter, encompasses the conserved DDE motif, D64-D116-E152 in HIV-1integrase. These residues are highly conserved in all integrases,retrotransposases and other DNA-processing enzymes (polynucleotidetransferases). Mutation of any of these acidic amino acids abolishesintegrase activities and viral replication. D64 and D116 are involved inthe formation of coordination complex with divalent metal (Mg2+ orMn2+). A second metal (Mg2+ or Mn2+) can be coordinated between D116 andE152 once HIV-1 integrase binds its DNA substrates (20, 21). Metal ioncoordination with viral integrase and the phosphodiester backbone of theDNA substrates are likely to occur during 3′-processing andstrand-transfer reactions. Region II is close to the active catalyticloop (amino acid residues 139-147), and involves the flexible loopformed by amino acid residues 140-149. This loop region has beenidentified as the DNA binding site which is important for integraseaction [19].

As seen in FIG. 17A, Ole binds to both regions I and II. Theβ-glycopyranose moiety of Ole interacts with residues in region IIwhereas the dihydroxyphenol ring occupies region I. FIGS. 17C and 17Dare ribbon representation of the HIV-integrase CCD showing major strongH-bonding sites with Ole and HT. The flexible loop widens the activesite region and allows the sugar ring of Ole to dock with strong H-bondwith P142 and Q148 as well as form weak interactions with S147. Thedihydroxyphenol moiety of Ole binds to region I with a strong H-bondinteraction with D64 and a weak H bond-network interactions with K156and K159. This suggests that Ole would be a strong integrase bindinginhibitor because it interacts with residues in both regions I and II.On the other hand, the dihydroxyphenol ring of HT binds to region IIwith strong H bond interaction with F139 and nearby T115, and weakinteractions with E138 and Q 148. Since the dihydroxyphenol ring iscapable of binding both regions I and II, HT maintains the ability tobind the integrase active site even if mutations occur. Thus thelikelihood of resistance development should be less than inhibitors thatbind to a single site. Thus, interaction modeling suggests that Ole andHT bind to both regions I and II, so they would be expected to beeffective against metal coordination as well as substrate binding.Modeling results therefore predict that Ole and HT would inhibit all ofthe three HIV-1 integrase activities.

Ole and HT Inhibit 3′-Processing Activity of HIV-1 Integrase

Modeling predictions that Ole and HT may affect HIV-integrase activitywere tested in all of the three activities, namely 3′-processing, strandtransfer (integration) and disintegration. Results of HIV-1 integraseinhibitory activities of Ole and HT are summarized in Table 7 with theiranti-HIV activities.

FIG. 18A shows the 3′-processing reaction and results. 5′ [³²P] labeled21-mer double-stranded oligonucleotide that mimics the U3HIV-1 LTR wasused as a substrate. 3′-processing by HIV-1 integrase, removes thedinucleotide GT from the 3′ end of the labeled minus strand of the21-mer substrate and yields a 3′ recessed product (U3-GT) with 19nucleotides in length. Ole or HT inhibits the 3′-processing activity ofHIV-1 integrase. FIG. 18A demonstrates dose-dependent inhibition of3′-processing by Ole and HT as detected by 7.5 M urea denaturatingpolyacrylamide gel electrophoresis. Inhibition of the formation of the19-mer product from the 21-mer substrate increases with the increase ofOle or HT concentration from 25 to 100 nM. The degree of inhibitiondepends on the concentrations of the HIV-1 integrase, the substrate andthe inhibitor. Under our assay conditions, EC₅₀s of 46 and 54 nM wereobtained for Ole and HT respectively, and total inhibition was observedat 100 nM. Substrate U5 HIV-LTR showed similar results.

Ole and HT Inhibit Strand-Transfer Activity of HIV-1 Integrase

The effect of Ole and HT on the strand-transfer activity of HIV-1integrase was tested by a quantitative heterologous integration assay.FIG. 18B shows the design and results of this assay: U3-GT, a 5′ [³²P]labeled and 3′-recessed 19-mer was used as the viral substrate. To focuson strand transfer, a supercoiled pUC18 plasmid DNA of 2.69 kb was usedas the heterologous target. Incubation of the 5′ [³²P] labeled viralsubstrate with unlabeled target in the presence of HIV-1 integraseresults in the integration of the labeled 19-mer viral substrate intothe 2.69 kb target plasmid. Integration was monitored by the conversionof unlabeled plasmid to labeled DNA in agarose gel electrophoresis asseen in the figure. Under these conditions, any inhibition detected mustbe specific for strand transfer and not for 3′ processing. Ole and HTdemonstrated dose-dependent inhibition of the strand-transfer activityof HIV-1 integrase with EC₅₀s of 56 and 43 nM respectively.

Ole and HT are Effective Against Disintegration Activity of HIV-1Integrase

FIG. 18C shows a schematic representation of disintegration reaction andassay results. Disintegration is the reverse of integration and involvesconcerted strand-cleavage and ligation reactions [20]. Strand cleavagetakes place precisely at the junction between the viral and the targetsequences and is coupled with the rejoining of the cleaved targetsequences. The disintegration substrate is a dumbbell shaped 38-meroligonucleotide that mimics the recombination intermediate of HIV-1integration. It contains 5′ [³²P] labeled virus-specific U5-LTR sequenceof 14-mer in the stem of the hairpin loop and arbitrary target DNAsequences of 24-mer in the base of the dumbbell [21,22]. The foldedstructure of the annealed substrate shown is based on reports obtainedfrom hairpin formation by similar sequences [23,24]. Dis-integration ofthe 5′ [³²P] labeled dumbbell by HIV-1 integrase is expected to give twoproducts, a 5′ [³²P] labeled 14-mer hairpin loop viral sequence and anunlabeled 24-mer closed circular target DNA. In the presence ofHIV-integrase, the production of labeled 14-mer hairpin loop wasdetected by 7.5 M urea denaturating polyacrylamide gel electrophoresisand autoradiography of the gel, whereas the 24-mer product, the targetDNA, was not seen in the autoradiography because it is unlabeled. Thisproduct can be detected by UV shadowing of the gel or by the use of3′-labeled substrate. In the presence of Ole or HT, dose-dependentinhibition on the formation of the labeled 14-mer disintegration productwas observed with EC₅₀s of 28 or 18 μM respectively.

TABLE 7 Anti-HIV Activity and Inhibition on HIV-1 Integrase ActivityAnti-HIV Activity^(a) Inhibitory EC₅₀ (nM) IC₅₀ (nM) Activity^(b) EC₅₀(nM) Syn- Cyto- HIV-1 Integrase Activities Compound cytium P24 toxicity3′-Proc ST Dis-In Ole 55 ± 5 73 ± 8 >10,000 46 ± 6 56 ± 5 28 ± 3 HT 61 ±6 68 ± 8 >10,000 54 ± 5 43 ± 5 18 ± 2 ^(a)EC₅₀, effective concentrationat 50% inhibition; IC₅₀, cytotoxicity concentration at 50% inhibition.^(b)3′-Proc, 3′-processing; ST, strand transfer; Dis-In, disintegration.Values are means ± SD of triplicates in three independent determinations

Discussion

Several classes of HIV integrase inhibitors have been reported [4,23],but none is clinically available yet. Lack of structural information forthe intact protein, issues regarding different active site conformationsdependent on crystal structure, and uncertain oligo-meric character ofthe enzyme protein have impeded the discovery of a clinically usefulHIV-integrase inhibitor [4,23]. Thus, molecular modeling becomes a keycomponent in both the design of new integrase inhibitors and theidentification of important protein-ligand interactions. There are 14crystal structures of HIV-integrase available from the Protein Data Bank(PDB); however, only one has an inhibitor bound in the active site: 1QS4[18]. We believe that this crystal structure contains the inhibitorwould be the most relevant active site conformation on which to conductthe docking simulations with our anti-HIV small molecules, Ole and HT,despite previously reported crystal-packing effects associated with thisstructure [24].

The docking results reported here show good correlation withexperimental data and provide a valuable tool for both evaluatingcompounds and designing more potent inhibitors. Ole and HT exhibit dosedependent inhibition in all of the three activities of HIV-1 integrase:3′-processing, strand transfer and disintegration with EC₅₀s all in thenM range. These compounds also showed good antiviral efficacy both incell-to-cell transmission of HIV-1 as assayed by syncytial formation andin HIV-1 replication as assayed by p24 production. However, they are nottoxic in the effective dose ranges and even at the concentration of1,000 times EC₅₀ (Table 7).

To our knowledge, Ole and HT are the first group of small moleculescapable of multiple actions against the AIDS virus, inhibiting bothviral entry and integration. To act both outside and inside of thecellular environments represents a great advantage of this novel classof drugs. The structure-function information described here shouldfacilitate the design of innovative multi-functional HIV-1 inhibitors.

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1. A method of modulating adipocyte differentiation or adipogenic geneor gene product expression or lipolytic gene or gene product expression,comprising administering a therapeutically effective amount ofoleuropein or an analogue, derivative, or oleuropein aglycone or theiranalogues, or derivatives thereof, or hydrotyrosol, or dihydroxy phenolor their analogues, or derivatives thereof, or elenolic acid or theiranalogues, or derivatives thereof, or an iridoid glycoside, or asecoiridoid glycoside or their analogue, derivative, or any combinationthereof including but not limited to olive leave extract to a mammaliansubject in need thereof.
 2. A method of treating, controlling orpreventing obesity, or of reducing body weight, or of inhibiting fataccumulation in vivo, or of promoting fat burning in vivo, comprisingadministering a therapeutically effective amount of oleuropein or ananalogue, derivative or oleuropein aglycone or their analogues, orderivatives thereof, or hydrotyrosol, or dihydroxy phenol or theiranalogues, or derivatives thereof, or elenolic acid or their analogues,or derivatives thereof, or an iridoid glycoside, or a secoiridoidglycoside or their analogue, derivative, or any combination thereofincluding but not limited to olive leave extract to a mammalian subjectin need thereof.
 3. A method of treating, controlling or preventing theonset of one or more obesity related disorders or conditions selectedfrom the group consisting of diabetes mellitus, hyperglycemia,hyperlipidemia, hypercholesteremia, atherosclerosis, hypertension,hypertriglyceridemia, insulin resistance, or hyperinsulinemia, in amammalian subject in need of treatment, comprising administering atherapeutically effective amount of oleuropein or an analogue,derivative or oleuropein aglycone or their analogues, or derivativesthereof, or hydrotyrosol, or dihydroxy phenol or their analogues, orderivatives thereof, or elenolic acid or their analogues, or derivativesthereof, or an iridoid glycoside, or a secoiridoid glycoside or theiranalogue, derivative, or any combination thereof including but notlimited to olive leave extract.
 4. A method of treating, controlling orpreventing the onset of one or more obesity related disorders orconditions selected from the group consisting of Asthma and relateddiseases including but not limited to, Allergy, Atopic Dermatitis(Eczema), Gastroesophageal Reflux Disease, Pneumothorax, Airway,Pulmonary and Lung disorders, Churg-Strauss Syndrome in a mammaliansubject in need of treatment, comprising administering a therapeuticallyeffective amount of oleuropein or an analogue, derivative or oleuropeinaglycone or their analogues, or derivatives thereof, or hydrotyrosol, ordihydroxy phenol or their analogues, or derivatives thereof, or elenolicacid or their analogues, or derivatives thereof, or an iridoidglycoside, or a secoiridoid glycoside or their analogue, derivative, orany combination thereof including but not limited to olive leaveextract.
 5. A method of treating, controlling or preventing the onset ofone or more obesity related disorders or conditions selected from thegroup consisting of AIDS and HAART related diseases including but notlimited to lipodystrophy in a mammalian subject in need of treatment,comprising administering a therapeutically effective amount ofoleuropein or an analogue, derivative or oleuropein aglycone or theiranalogues, or derivatives thereof, or hydrotyrosol, or dihydroxy phenolor their analogues, or derivatives thereof, or elenolic acid or theiranalogues, or derivatives thereof, or an iridoid glycoside, or asecoiridoid glycoside or their analogue, derivative, or any combinationthereof including but not limited to olive leave extract.
 6. A method oftreating, controlling or preventing the onset of one or more viralinfection related disorders or conditions selected from the groupconsisting of viral infection related diseases including but not limitedto the AIDS virus HIV-1, and other viruses with type I transmembraneenvelope glycoprotein, such as simian immunodeficiency viruses (SIV),Sendai virus, feline immunodeficiency virus (FIV), respiratory syncytialvirus (RSV), measles virus, Ebola virus, Nipah and Hendra viruses, thesevere acute respiratory syndrome associated coronavirus (SARS-CoV), andthe avain flu virus in mammalian, avian, poultry, non human primatessubjects in need of treatment, comprising administering atherapeutically effective amount of oleuropein or an analogue,derivative or oleuropein aglycone or their analogues, or derivativesthereof, or hydrotyrosol, or dihydroxy phenol or their analogues, orderivatives thereof, or elenolic acid or their analogues, or derivativesthereof, or an iridoid glycoside, or a secoiridoid glycoside or theiranalogue, derivative, or any combination thereof including but notlimited to olive leave extract.
 7. The method of claim 1, wherein theadipogenic genes and gene products whose expression is modulatedincludes, but is not limited to, PPARγ, PPARγ2, LPL and the αP2 genesand gene products.
 8. The method of claim 1, wherein the lipolytic genesand gene products whose expression is modulated including but notlimited to PPAR δ and its modulated genes and gene products.
 9. Themethod of claim 1, wherein the lipogenic or lipolytic genes whoseexpression is modulated including but not limited to PPAR α and itsmodulated genes and gene products.
 10. A method of de-differentiationand transdifferentiating adipocytes into osteoblasts (bone cells),myoblasts (muscle cells), and chondrocytes (cartilage cells), the methodcomprising administering to a mammal in need thereof a therapeuticallyeffective amount of oleuropein or an analogue, derivative or oleuropeinaglycone or their analogues, or derivatives thereof, or hydrotyrosol, ordihydroxy phenol or their analogues, or derivatives thereof, or elenolicacid or their analogues, or derivatives thereof, or an iridoidglycoside, or a secoiridoid glycoside or their analogue, derivative, orany combination thereof including but not limited to olive leaveextract.
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 18. The method of claim 2further comprising administering a second agent in combination witholeuropein, or an analogue or oleuropein aglycone or their analogues, orderivatives thereof, or hydrotyrosol, or dihydroxy phenol or theiranalogues, or derivatives thereof, or elenolic acid or their analogues,or derivatives thereof, or an iridoid glycoside, or a secoiridoidglycoside or their analogue, derivative, or any combination thereofincluding but not limited to olive leave extract.
 19. The method ofclaim 18, wherein the second agent is selected from the group consistingof a different PPAR modulating agent, a cholesterol or lipid loweringagent, a biguanide, insulin, an antihyperglycemic agent and any agentuseful for treating metabolic syndrome or type 2 diabetes.
 20. Themethod of claim 19, wherein the different PPAR modulating agent isselected from the group consisting of troglitazone, pioglitazone androsiglitazone.
 21. The method of claim 19, wherein the cholesterol orlipid lowering agent is a HMG-CoA reductase inhibitor, and wherein theHMG-CoA reductase inhibitor is a statin selected from the groupconsisting of atorvastatin, bervastatin, cerivastatin, dalvastatin,fluvastatin, itavastatin, lovastatin, mevastatin, nicostatin,nivastatin, pravastatin and simvastatin.
 22. The method of claim 19,wherein the biguanide is selected from the group consisting ofmetformin, phenformin, and buformin.
 23. The method of claim 19, whereinthe antihyperglycemic is a prandial glucose regulator or analpha-glucosidase inhibitor.
 24. The method of claim 23, wherein theprandial glucose regulator is repaglinide or nateglinide.
 25. The methodof claim 23, wherein the alpha-glucosidase inhibitor is selected fromthe group consisting of acarbose, voglibose and miglitol.
 26. The methodof claim 19, wherein the agent useful for treating metabolic syndrome ortype 2 diabetes is a sulfonylurea selected from the group consisting ofglimepiride, glibenclamide (glyburide), gliclazide, glipizide,gliquidone, chloropropamide, tolbutamide, acetohexamide, glycopyramide,carbutamide, glibonuride, glisoxepid, glybuthiazole, glibuzole,glyhexamide, glymidine, glypinamide, phenbutamide, tolcylamide andtolazamide.
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